FIXaxFX bispecific antibody with common light chain

ABSTRACT

Bispecific antigen binding molecules (e.g., antibodies) that bind blood clotting factors, factor IXa (FIXa) and factor X (FX), and enhance the FIXa-catalysed activation of FX to FXa. Use of the bispecific antigen binding molecules to control bleeding, by replacing natural cofactor FVIIIa which is deficient in patients with haemophilia A.

FIELD OF THE INVENTION

This invention relates to bispecific antigen-binding molecules (e.g.,antibodies) that bind factor IXa and factor X clotting factors in theblood coagulation cascade. Such bispecifics functionally substitute forfactor VIII by activating factor X, restoring blood clotting ability topatients who are deficient in FVIII, i.e., patients who have type Ahaemophilia.

BACKGROUND

Haemophilia is an inherited condition in which the blood has a reducedability to clot, owing to loss of function (partial or total) of one ofthe many clotting factors. Haemophilia A is a deficiency in bloodclotting factor VIII (FVIII). The disease has mild, moderate and severeforms, depending on the degree to which the patient retains any residualFVIII function and on the balance of other components in the bloodcoagulation cascade. If untreated, haemophilia A leads to uncontrolledbleeding, which can result in severe disability, especially throughdamage to joints from haemarthrosis events. The disease is oftenlife-limiting and can be life-threatening. The global incidence ofhaemophilia A is believed to be around 1:10,000. Haemophilia B(deficiency of a different blood clotting factor, factor IX) is lesscommon, with an incidence of around 1:50,000. Both diseases are X-linkedso are usually found in males, the incidence of haemophilia A in malebirths thus being around 1 in 5,000.

Preventing bleeding episodes is essential to improving patients' qualityof life and reducing the risk of fatal blood loss. For haemophilia A,the missing co-factor can be replaced by administration of FVIII. FVIIIfor administration to a patient may be recombinantly expressed or it maybe purified from blood plasma. Typically, patients on this treatmentself-inject with FVIII every 48 hours or 3× per week.

Treatment with FVIII is not a perfect solution. A serious drawback isthat it can trigger production of allo-antibodies in the body. Thisrenders treatment with FVIII ineffective, as the allo-antibodies bindthe FVIII and prevent its activity, putting the patient in a dangeroussituation if a bleed occurs. Such inhibitory antibodies develop in about30% of patients treated with FVIII for severe haemophilia.

Treatment with plasma-derived FVIII, rather than the recombinant form,has been reported to have a lower risk of triggering inhibitoryantibodies in patients. This may be due to the plasma-derived formretaining Von Willebrand factor (VWF), which is found naturally inassociation with FVIII and may mask immunogenic epitopes. However, noform of FVIII has yet been produced that completely avoids the risk ofinhibitory antibodies.

Despite being possibly more immunogenic, recombinant FVIII offers someadvantages over the plasma-derived form, since being more stable it iseasier and cheaper to store and transport. The risk of transmittinginfections via products from donated blood plasma is now much reducedcompared with the 1980s when viruses such as hepatitis C and HIV wereinadvertently spread to recipients of infected blood products, but ofcourse the need for strict safety controls remains.

New recombinant forms of FVIII have been developed, such as the B-domaintruncated polypeptide turoctocog alfa (NovoEight®). However, suchproducts are ineffective for patients that develop neutralisingantibodies against FVIII. Some patients successfully undergo immunetolerance induction to prevent anti-FVIII antibodies from developing.However, there remains a substantial demand for alternatives to FVIIIfor use in patients who have, or are at risk of developing, inhibitoryantibodies.

One such alternative is recombinant factor VIIa, known as activatedeptacog alfa (NovoSeven®). However, it has a short half-life and must beinjected every few hours. Its use is largely restricted to rescuetherapy or providing haemostatic cover during surgery in haemophiliacswho have inhibitory antibodies, rather than being a viable option forlong term protective treatment.

Another available product is FEIBA (Factor Eight Inhibitor BypassingActivity), an activated prothrombin complex concentrate (aPCC), whichsimilarly can be used to control bleeding episodes and to preventbleeding during surgical interventions in haemophiliac patients who haveinhibitors to factor VIII.

A variety of other alternative therapies are currently being pursued,such as gene therapy, suppression of anti-thrombin using siRNA, and anantibody to TFPI (Tissue factor Pathway Inhibitor), concizumab.

One approach is a humanised bispecific IgG antibody targeting bothfactor IXa (FIXa) and factor X (FX). The bispecific antibody binds FIXawith one arm and FX with the other arm, bringing these two co-factorstogether and thereby promoting FIXa-catalysed activation of FX in thesame way that FVIII does. Thus, the antibody functionally replaces FVIIIin the blood coagulation cascade (FIG. 1). As its structure iscompletely different from FVIII, the antibody cannot be neutralised byanti-FVIII antibodies and so is suitable for patients who havedeveloped, or are at risk of developing, allo-antibodies to administeredFVIII.

In 2012, Kitazawa et al reported isolation of a FIXa/X bispecificantibody which was able to activate FX, from a screen of approximately40,000 anti-FIXa/X bispecific antibodies that had been produced byimmunising 92 laboratory animals with human FIXa or FX andco-transfecting the anti-FIXa and anti-FX antibody genes into host cellsfor expression [1]. The selected antibody was refined to generate ahumanised antibody designated hBS23, which showed coagulation activityin FVIII-deficient plasma and in vivo haemostatic activity in primates[1]. A more potent version of this antibody, designated hBS910 [2],entered clinical trials under the investigational drug name ACE910, INNemicizumab [3]. The development of ACE910 took place in one of theleading antibody groups globally. Nevertheless, it took more than 7years to engineer a molecule with the appropriate in vivo efficacy andwith biochemical and biophysical properties suitable for clinical scalemanufacturing.

In a phase I study of 48 healthy male subjects receiving ACE910subcutaneously at doses up to 1 mg/kg, 2 subjects tested positive foranti-ACE910 antibodies [4]. The antibody was reported to have a linearpharmacokinetic profile and a half-life of about 4-5 weeks [4].Emicizumab was subsequently administered to 18 Japanese patients withsevere haemophilia A, at weekly subcutaneous doses of up to 3 mg/kg, andwas reported to reduce the episodic use of clotting factors to controlbleeding in these patients [5]. In December 2016, emicizumab wasreported to have met its primary endpoint in a phase III clinical trialfor reducing bleeding in patients with haemophilia A (the “HAVEN 1”study). A statistically significant reduction in the number of bleedswas reported for patients treated with emicizumab prophylaxis comparedwith those receiving no prophylactic treatment. The study was alsoreported to have met all secondary endpoints, including a statisticallysignificant reduction in the number of bleeds over time with emicizumabprophylaxis treatment in an intra-patient comparison in people who hadreceived prior bypassing agent prophylaxis treatment. The efficacy dataon emicizumab are therefore encouraging, although safety concerns wereheightened by the death of a patient on the HAVEN 1 study. The approveddrug carries a boxed warning regarding the risk of thromboticmicroangiopathy and thromboembolism in patients receiving aPCC incombination with emicizumab. As noted above, aPCC is used to controlbleeding in patients who have inhibitory antibodies to FVIII, a keypatient group for treatment with the bispecific antibody.

It is important to note that management of haemophilia requirescontinuous treatment for a patient's lifetime, beginning at the point ofdiagnosis—which is usually in infancy—and calls for a therapy that willbe tolerated without adverse effects and that will remain effective overseveral decades or even a century. Long term safety, including lowimmunogenicity, is therefore of greater significance for ananti-haemophilia antibody compared with antibodies that are intended tobe administered over a shorter duration such as a period of weeks,months or even a few years.

WO2018/098363 described bispecific antibodies binding to FIX and FX,isolated from a human antibody yeast library (Adimab). WO2018/098363disclosed that increasing the affinity of the anti-FIXa arm of abispecific antibody results in an increase in FVIIIa activity(represented by decreased blood clotting time in an assay). A bispecificantibody “BS-027125” was generated by affinity maturation of aninitially selected “parent” antibody, which increased the affinity ofits FIXa-binding arm. BS-027125 was reported to achieve approximately90% FVIIIa-like activity in a one-stage clotting assay. When comparedwith emicizumab, BS-027125 was reported to exhibit much higher affinitybinding to factor FIX zymogen, FIXa and FX zymogen, and much lowerbinding (no detected binding) to FXa. The FIX-binding arm, “BIIB-9-1336”reportedly showed selective binding for FIXa (activated FIX) inpreference to FIX zymogen (mature FIX prior to proteolytic activation),and was found to bind an epitope overlapping with the FIXa epitope boundby FVIIIa. The FX-binding arm, “BIIB-12-917”, reportedly showedselective binding to FX zymogen, lacked detectable binding to(activated) FXa, and bound an epitope of FX that lies within theactivation peptide (which is present in FX zymogen but not FXa). Furthermutations were then introduced into selected FIX-binding antibodies,including BIIB-9-1336, to generate libraries from which to select forantibodies with even further increased specificity and/or affinity forFIXa.

WO2018/141863 and WO2018/145125 also described anti-FIXaxFX bispecificantibodies and their use as procoagulants for treating or reducingbleeding.

SUMMARY OF THE INVENTION

The present invention relates to improved bispecific antigen-bindingmolecules that bind blood clotting factors FIXa and FX. The bispecificantigen-binding molecules of the present invention enhance theFIXa-catalysed activation of FX to FXa, and can effectively replace thenatural cofactor FVIIIa which is missing in patients with haemophilia A,to restore the ability of the patients' blood to clot. See FIG. 2.

As reported here, the inventors succeeded in generating a number ofbispecific antigen-binding molecules having suitable qualities fordevelopment as therapeutic products, including very high potency inenhancing FX activation. Described are bispecific antigen-bindingmolecules having novel binding sites for anti-FIXa and anti-FX, whichcan be used to effectively substitute for FVIIIa in the blood clottingcascade. In particular, an anti-FIXa binding site is described which ishighly active in combination with an array of different anti-FX bindingsites and can thus be incorporated into a variety of different FIXa-FXbispecifics, providing flexibility for selection of bispecificantibodies with further desired characteristics such as ease ofmanufacture.

The inventors have designed bispecific antibodies which combine a potentFVIII mimetic activity (as indicated by high performance in in vitroassays) with robust biochemical and biophysical properties suitable forclinical scale manufacturing (including expression, bispecific molecularassembly, purification and formulation), and which are of fully humanorigin, thereby minimising the risk of immunogenicity in human in vivotherapy.

Aspects of the invention are set out in the appended claims, and furtherembodiments and preferred features of the invention are described below.

In a first aspect, the present invention relates to bispecificantigen-binding molecules comprising (i) a FIXa binding polypeptide armcomprising a FIXa binding site, and (ii) a FX binding polypeptide armcomprising a FX binding site. The FIXa and/or the FX binding polypeptidearm may comprise an antibody Fv region comprising the FIXa or FX bindingsite respectively. An antibody Fv region is an antibody VH-VL domainpair. The VH domain comprises HCDR1, HCDR2 and HCDR3 in a VH domainframework, and the VL domain comprises LCDR1, LCDR2 and LCDR3 in a VLdomain framework. The polypeptide arm may comprise an antibody heavychain (optionally one comprising an IgG constant region) and/or anantibody light chain.

Antigen-binding molecules of the present invention may thus comprise

-   -   first and second antibody Fv regions, the first and second        antibody Fv regions comprising binding sites for FIXa and for FX        respectively, and    -   a half-life extending region for prolonging the half-life of the        molecule in vivo.

The half-life extending region may be a heterodimerisation region,comprising a first polypeptide covalently linked (e.g., as a fusionprotein) to the first antibody Fv region and a second polypeptidecovalently linked (e.g., as a fusion protein) to the second antibody Fvregion, wherein the two polypeptides pair covalently and/ornon-covalently with one another. The first and second polypeptides ofthe heterodimerisation region may have identical or different amino acidsequences. The heterodimerisation region may comprise one or moreantibody constant domains, e.g., it may be an antibody Fc region.

Bispecific antigen-binding molecules of the present invention are ableto bind FIXa through the FIXa binding site of the FIXa bindingpolypeptide arm and to bind FX through the FX binding site of the FXbinding polypeptide arm, and thereby enhance the FIXa-catalysedactivation of FX to FXa. This may be determined in an in vitro FXactivation assay as described herein.

The FIXa binding site may be provided by a set of complementaritydetermining regions (CDRs) in the FIXa binding polypeptide arm, the setof CDRs comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3.Optionally, HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3is SEQ ID NO: 408. Optionally, LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ IDNO: 7 and LCDR3 is SEQ ID NO: 8.

The set of HCDRs in the FIXa binding polypeptide arm may be the set ofHCDRs of any anti-FIX VH domain shown herein, such as any shown in TableS-9A, any identified in Table N, or any of the VH domains N0128H,N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H or N1333H shownin FIG. 20. HCDR1 may be SEQ ID NO: 441. HCDR2 may be SEQ ID NO: 634 orSEQ ID NO: 436. HCDR3 may be SEQ ID NO: 635 or SEQ ID NO: 433. The CDRsmay be the N1280 CDRs, wherein HCDR1 is SEQ ID NO: 441, HCDR2 is SEQ IDNO: 436 and HCDR3 is SEQ ID NO: 533. Alternatively the CDRs may be theN1333H CDRs.

The set of LCDRs in the FIXa binding polypeptide arm may be the set ofLCDRs of any anti-FIX VL domain shown herein. The LCDRs may be the LCDRsof 0128L as shown in Table S-50. LCDR1 may be SEQ ID NO: 6, LCDR2 may beSEQ ID NO: 7 and/or LCDR3 may be SEQ ID NO: 8.

Optionally, one or more amino acids in the set of CDRs may be mutated todiffer from these sequences. For example, the set of CDRs may comprise1, 2, 3, 4 or 5 amino acid alterations, the altered residue or residuesbeing in any one or more of the heavy or light chain CDRs. For examplethe set of CDRs may comprise one or two conservative substitutions. Thechoice of mutations, e.g., substitutions, can be informed by theinformation and analysis provided in the Examples herein.

The FIXa binding polypeptide arm may comprise an antibody VH domaincomprising a set of HCDRs HCDR1, HCDR2 and HCDR3. The sequence of HCDR1may be SEQ ID NO: 406, optionally with one or two amino acid alterations(e.g., substitutions). The sequence of HCDR2 may be SEQ ID NO: 407,optionally with one or two amino acid alterations (e.g., substitutions).The sequence of HCDR3 may be SEQ ID NO: 408, optionally with one or twoamino acid alterations (e.g., substitutions).

The FIXa binding polypeptide arm may comprise an antibody VL domaincomprising a set of LCDRs LCDR1, LCDR2 and LCDR3. The sequence of LCDR1may be SEQ ID NO: 6, optionally with one or two amino acid alterations(e.g., substitutions). The sequence of LCDR2 may be SEQ ID NO: 7,optionally with one or two amino acid alterations (e.g., substitutions).The sequence of LCDR3 may be SEQ ID NO: 8, optionally with one or twoamino acid alterations (e.g., substitutions).

The antibody Fv region of the FIXa binding polypeptide arm may comprisea VH domain generated through recombination of immunoglobulin heavychain v, d and j gene segments, wherein the v gene segment is VH3-7(e.g., VH3-7*01), wherein the j gene segment is JH6 (e.g. JH6*02), andoptionally wherein the d gene segment is DH1-26 (e.g., DH1-26*01),and/or it may comprise a VL domain generated through recombination ofimmunoglobulin light chain v and j gene segments, wherein the v genesegment is VL3-21 (e.g., VL3-21*d01) and the j gene segment is JL2(e.g., JL2*01). In another embodiment, a VL domain may be one that isgenerated through recombination of immunoglobulin light chain v and jgene segments, wherein the v gene segment is VL3-21 (e.g., VL3-21*d01)and the j gene segment is JL3 (e.g., JL3*02).

The amino acid sequence of the VH domain of a FIXa polypeptide bindingarm may share at least 90% sequence identity with a VH domain shown inFIG. 20, e.g., the N1280H VH domain. Sequence identity may be at least95%, at least 97%, at least 98% or at least 99%. Optionally the VHdomain is one of the anti-FIX VH domains shown herein, such as any shownin Table S-9A, any identified in Table N, or any of the VH domainsN0128H, N0436H, N0511H, N1091H, N1172H, N1280H, N1314H, N1327H or N1333Hshown in FIG. 20. Optionally the VH domain is N1280H, N1333H, N1441,N1442 or N1454. Optionally the anti-FIXa VH domain comprises the aminoacid sequence of any of said VH domains (e.g., N1280H) with up to 5amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions.Substitutions may optionally be in one or more framework regions, e.g.,there may be 1 or 2 substitutions in FR3, optionally at IMGT position 84and/or IMGT position 86.

The amino acid sequence of the VL domain may share at least 90% sequenceidentity with SEQ ID NO: 10 (0128L). Sequence identity may be at least95%, at least 96%, at least 97%, at least 98% or at least 99%.Optionally the VL domain amino acid sequence is SEQ ID NO: 10. The VLdomain amino acid sequence may alternatively be SEQ ID NO: 416.

The FX binding site may be provided by a set of CDRs in the FX bindingpolypeptide arm. The FX binding polypeptide arm may comprise an antibodyVH-VL domain pair (i.e., an antibody Fv region), the VH domaincomprising HCDR1, HCDR2 and HCDR3 in a framework, and the VL domaincomprising LCDR1, LCDR2 and LCDR3 in a framework.

The FX binding site may be provided by the HCDRs of any anti-FX VHdomain identified herein (e.g., any set of HCDR1, HCDR2 and HCDR3 of aVH domain shown in Table S10-C and/or in FIG. 11) and the 0128L LCDRs.

The FX binding polypeptide arm may comprise a VH domain having at least90% amino acid sequence identity with a VH domain disclosed herein,including any in Table S-10C and/or in FIG. 11—for example the T0687H,T0736H or T0999H VH domain. Sequence identity may be at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%. Optionally the VHdomain comprises the amino acid sequence of said VH domain with up to 5amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions.Substitutions may optionally be in one or more framework regions.

The FX binding polypeptide arm may comprise a VH domain having at least90% amino acid sequence identity with the T0201 VH domain (shown in FIG.11). Sequence identity may be at least 95%, at least 96%, at least 97%,at least 98% or at least 99%. Optionally the VH domain comprises theamino acid sequence of said VH domain with up to 5 amino acidsubstitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Substitutions mayoptionally be in one or more framework regions.

The FX binding polypeptide arm may comprise any VH domain amino acidsequence identified herein, such as any shown in Table S-10C, anyidentified in Table T or any from FIG. 11. Optionally the VH domain isT0201 H, T0687H, T0736H or T0999H.

The FX binding polypeptide arm may comprise a VL domain having at least90% amino acid sequence identity with the 0128L VL domain SEQ ID NO: 10.Sequence identity may be at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%. Optionally the VL domain comprises the aminoacid sequence of the 0128L VL domain with up to 5 amino acidsubstitutions, i.e., 1, 2, 3, 4 or 5 substitutions. Optionally the VLdomain amino acid sequence is SEQ ID NO: 10. Alternatively the VL domainsequence is SEQ ID NO: 416.

The FX binding polypeptide arm may comprise an antibody Fv regioncomprising

-   -   a VH domain generated through recombination of immunoglobulin        heavy chain v, d and j gene segments, wherein the v and j gene        segments are IGHV1-46 (e.g., VH1-46*03) and IGHJ1 (e.g.,        JH1*01), and optionally wherein the d gene segment is IGHD6-6        (e.g., DH6-6*01), and    -   a VL domain generated through recombination of immunoglobulin        light chain v and j gene segments, wherein the v and j gene        segments are IGLV3-21 (e.g., VL3-21*d01) and IGLJ2 (e.g.,        JL2*01) or IGLJ3 (e.g., JL3*02).

Accordingly, one aspect of the present invention is a bispecificantibody that binds FIXa and FX and catalyses FIXa-mediated activationof FX, wherein the antibody comprises two immunoglobulin heavy-lightchain pairs, wherein

-   -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain comprises a set of HCDRs comprising HCDR1,        HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1        is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID        NO: 408, and/or wherein the first VH domain is at least 95        identical to the N1280H VH domain at the amino acid sequence        level;    -   the second VH domain is at least 95% identical to the T0201H VH        domain at the amino acid sequence level, and    -   the first VL domain and the second VL domain each comprise a set        of LCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid        sequences defined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID        NO: 7 and LCDR3 is SEQ ID NO: 8, and/or wherein the first VL        domain and the second VL domain are at least 95% identical to        the 0128L VL domain SEQ ID NO: 10 at the amino acid sequence        level.

Another aspect of the present invention is a bispecific antibody thatbinds FIXa and FX and catalyses FIXa-mediated activation of FX, whereinthe antibody comprises two immunoglobulin heavy-light chain pairs,wherein

-   -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain is a product of recombination of human        immunoglobulin heavy chain v, d and j gene segments, wherein the        v gene segment is IGHV3-7 (e.g., VH3-7*01) and the j gene        segment is IGHJ6 (e.g., JH6*02),    -   the second VH domain is a product of recombination of human        immunoglobulin heavy chain v, d and j gene segments, wherein the        v gene segment is IGHV1-46 (e.g., VH1-46*03) and the j gene        segment is IGHJ1 (e.g., JH1*01), and optionally wherein the d        gene segment is IGHD6-6 (e.g., DH6-6*01), and    -   the first VL domain and the second VL domain are both products        of recombination of human immunoglobulin light chain v and j        gene segments, wherein the v gene segment is IGLV3-21 (e.g.,        VL3-21*d01) and the j gene segment is IGLJ2 (e.g., JL2*01) or        IGLJ3 (e.g., JL3*02).

Another aspect of the present invention is a bispecific antibody thatbinds FIXa and FX and catalyses FIXa-mediated activation of FX, whereinthe antibody comprises two immunoglobulin heavy-light chain pairs,wherein

-   -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain has at least 95% amino acid sequence        identity with the N1280H VH domain,    -   the second VH domain has at least 95% amino acid sequence        identity with the T0201H VH domain, and    -   the first VL domain and the second VL domain each have at least        95% amino acid sequence identity with the 0128L VL domain.

The first VH domain may comprise a set of HCDRs comprising HCDR1, HCDR2and HCDR3 with amino acid sequences defined wherein HCDR1 is SEQ ID NO:406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID NO: 408.

The first VH domain may have at least 96%, at least 97%, at least 98% orat least 99% amino acid sequence identity to N1280H. The first VH domainmay comprise a set of N1280H HCDRs comprising N1280H HCDR1, N1280H HCDR2and N1280H HCDR3. For example, it may be the N1280H VH domain.Alternatively, the VH domain may be the N1441H, N1442H or N1454H VHdomain.

Amino acid sequences of example VH domains and sets of VH CDRs are shownin FIG. 20 and/or in Table S-9A. The first VH domain of the bispecificantibody may be, or may have at least 95%, at least 96%, at least 97%,at least 98% or at least 99% amino acid sequence identity to, any ofthese VH domains. Optionally it may comprise a VH domain amino acidsequence having up to 5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5substitutions compared with said VH domain. Substitutions are optionallyin framework regions.

Examples of residues and substitutions that may be retained orintroduced in the first VH domain include the following (defined withreference to N1280H, with IMGT numbering as shown in FIG. 14):

Substitution of another residue (e.g., Asp, Glu, His, Asn, Gln, Met,Thr, Gly, Ser, Ala, Ile, Leu, Val or Tyr) at Lys84 in FR3, e.g.,Lys84Asp or Lys84Glu; and Substitution of another residue at Ser86 inFR3, e.g., a negatively charged residue such as Glu (Ser86Glu).

Further examples include:

Substitution of a negatively charged residue (e.g., Asp or Glu) or Hisat one or more of Gln3, Val5, Gly9, Gly11, Gly16, Gly17 and Leu21 FR1,such as any of Gln3Asp, Gln3Glu, Gln3His, Val5Glu, Gly9Glu, Gly11Asp,Gly11Glu, Gly11His, Gly16Glu, Gly17Asp, Gly17Glu or Leu21Asp;

Substitution of a negatively charged residue at Val68 and/or Val71 inFR3, e.g., Val68Asp, Val68Glu or Val71Glu;

Substitution of His, Gln or Leu at Arg75 in FR3;

Substitution of Ser, Thr, Gly, Leu or Lys at Arg80 in FR3; and

Substitution of Asp or His at Asn82 in FR3.

Any one or more of the above-listed sequence features may be included.

The second VH domain may be, or may have at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% amino acid sequence identity toT0201H or any other VH domain shown in FIG. 11 and/or in Table S-10C.Optionally it may comprise a VH domain amino acid sequence having up to5 amino acid substitutions, i.e., 1, 2, 3, 4 or 5 substitutions comparedwith said VH domain. Substitutions are optionally in framework regions.The second VH domain may comprise an HCDR1 which is the T0201H HCDR1, anHCDR2 which is the T0201H HCDR1, and/or an HCDR3 which is the T0201HHCDR3. Amino acid sequences of these CDRs are shown in FIGS. 11 and 12and in Table S-10C. Further example CDRs are indicated in FIG. 12 and inTable T. For example, the second VH domain may comprise:

-   -   an HCDR1 which is the T0201 HCDR1 or the T0736 HCDR1,    -   an HCDR2 which is the T0201 HCDR2, and/or    -   an HCDR3 which is the T0201 HCDR3, the T0687 HCDR3 or the T0736        HCDR3.

Optionally, HCDR1 is SEQ ID NO: 636 or SEQ ID NO: 598. Optionally, HCDR2is SEQ ID NO: 467. Optionally, HCDR3 is SEQ ID NO: 637, SEQ ID NO: 638,SEQ ID NO: 639 or SEQ ID NO: 565.

Examples of residues and substitutions that may be retained orintroduced in the second VH domain include the following (defined withreference to T0201H, with IMGT numbering as shown in FIG. 13):

-   -   Substitution of another amino acid residue at Cys114, e.g.,        wherein the substituted residue is Ile, Gln, Arg, Val or Trp,        (preferably Ile, Val or Leu);    -   Substitution of another positively charged residue for Gln3 in        FR1, e.g., Gln3Arg or Gln3Lys;    -   Germlining of residues in framework regions, e.g., Ile5Val        (substitution of valine for isoleucine at residue 5 in FR1), or        replacement of a non-germline residue in a framework region by a        different non-germline residue, e.g., Ile5Arg (substitution of        arginine for isoleucine at residue 5 in FR1);    -   Substitution of another amino acid residue (e.g., Lys, Ala or        Gly) at Glu11 in FR1, e.g., Glu11Lys;    -   Substitution of a positively charged amino acid residue for        Gly16 in FR1, e.g., Gly16Arg;    -   Presence of Met at position 39 in FR2, or alternatively Leu at        this position;    -   Presence of Ser at position 62 and/or position 64 in CDR2;    -   Substitution of Tyr at Phe71 in FR3;    -   Substitution of a positively charged residue at Thr82 in FR3,        e.g., Thr82Arg or Thr82Lys; Presence of Ser at position 85 in        FR3, or alternatively Thr at this position;

Substitution of a positively charged residue at Thr86 in FR3, e.g.,Thr86Arg or Thr86Lys.

Any one or more of the above-listed sequence features may be included.

The FIXa binding polypeptide arm and the FX binding polypeptide arm mayeach comprise an antibody Fv, wherein the VL domain of each Fv has anidentical amino acid sequence, i.e. the bispecific antigen-bindingmolecule has a common VL domain. The molecule may have a common lightchain comprising a variable region and a constant region, optionally ahuman lambda constant region.

The bispecific antigen-binding molecule may be a tetramericimmunoglobulin comprising

-   -   a first pair of antibody heavy and light chains (heavy-light        chain pair) comprising a FIXa binding Fv region,    -   a second heavy-light chain pair comprising a FX binding Fv        region,    -   wherein each heavy chain comprises a VH domain and a constant        region, and each light chain comprises a VL domain and a        constant region, and wherein the first and second heavy-light        chain pairs associate through heterodimerisation of their heavy        chain constant regions to form the immunoglobulin tetramer.

As noted, the light chain may be a common light chain, i.e., the lightchain of the first and second heavy-light chain pairs has an identicalamino acid sequence. Each heavy-light chain pair may comprise the 0128LCL constant domain paired with a CH1 domain. The sequence of the lightchain may be SEQ ID NO: 405. Alternatively the sequence of the lightchain may be SEQ ID NO: 414. Exemplary immunoglobulin isotypes includehuman IgG, e.g., IgG4, optionally with engineered constant domains suchas IgG4 PE.

The Fc domain of a bispecific antibody may be engineered to promoteheterodimerisation over homodimerisation. For example, the heavy chainconstant region of the first heavy-light chain pair may comprise adifferent amino acid sequence from the heavy chain constant region ofthe second heavy-light chain pair, wherein the different amino acidsequences are engineered to promote heterodimerisation of the heavychain constant regions. Examples include knobs-into-holes mutations orcharge pair mutations. Alternatively, the heavy chain constant region ofthe first heavy-light chain pair may be identical to the heavy chainconstant region of the second heavy-light chain pair, in which case itis expected that both homodimers and heterodimers will assemble, andthese will be subsequently separated using one or more purificationsteps in the antibody manufacturing process to isolate the desiredheterodimer comprising one anti-FIXa arm and one anti-FX arm.

An advantageous feature of bispecific antibodies exemplified here isthat they have been generated from human immunoglobulin gene segments,using the Kymouse platform. Unlike antibodies generated fromimmunisation of normal laboratory animals, which may require“humanisation” steps such as grafting of mouse CDRs into human antibodyvariable domains and iterative refinement of the engineered variabledomains to mitigate a loss of function resulting from these changes, theantibodies of the present invention were generated and selected from theoutset with fully human antibody variable domains. The use of a fullyhuman antibody is of special relevance in the context of haemophiliatreatment, where low immunogenicity is paramount, as noted above. Thelow immunogenicity of the bispecific antibodies of the present inventionrenders them suitable for treatment of haemophilia A patients, includingthose with or without inhibitory antibodies to other treatments such asFVIII. Patients receiving antigen-binding molecules of the presentinvention should be at minimal risk of developing an immunogenicresponse to the therapy.

The mode of action of the bispecific molecules is also associated with agood safety profile, with low risk of complications such as deep veinthrombosis and pulmonary embolism. Activity of the bispecific moleculesis comparable with that of natural FVIII and a mechanism of action thatis integrated within the existing blood coagulation pathway, beingactivated only in the context of upstream triggering of the naturalclotting cascade.

Bispecific antibodies according to the present invention have shownstrong activity in a number of functionally relevant assays for FVIIImimetic activity, including factor Xase assay, activated partialthromboplastin time (aPTT) assay and thrombin generation assay (TGA), asexemplified herein.

Other desirable features include long-half life (reducing the requiredfrequency of administration) and amenability of the molecules toformulation at high concentration (facilitating subcutaneous injectionin the home setting).

Patient compliance is recognised to be a significant issue for long termself-administered therapy, especially among teenage and young adultpatients. For a treatment to succeed in the field, its administrationschedule should be simple for the patient to understand and follow withminimum inconvenience. Long intervals between administered doses aredesirable, but reducing dose frequency without sacrificing therapeuticactivity requires a product with both a long in vivo half life and asufficient efficacy at “trough” concentrations towards the end of adosing period. Antigen-binding molecules according to the presentinvention desirably have a long in vivo half life. This can befacilitated by inclusion of an Fc region which undergoes recycling invivo via FcRn. Antigen-binding molecules according to the presentinvention also preferably maintain high functional activity at lowconcentration. We found that bispecific antibodies according to thepresent invention have a thrombogenic activity similar to that ofemicizumab but with an increase in thrombogenic activity that is mostpronounced at lower concentrations. Data disclosed herein indicate thatbispecific antibodies according to the present invention possess athrombogenic activity that is the same as or surpasses that ofemicizumab at concentrations in at least the range of 1 to 300 nM, forexample when the antibody and emicizumab are tested at the followingconcentrations:

-   -   1-30 nM, e.g., at 1 nM, at 3 nM, at 10 nM and/or at 30 nM;    -   100-300 nM, e.g., at 100 nM and/or at 300 nM.

Activity can be measured in the thrombin generation assay describedherein. Effective activity at low concentrations may help to ensure thatprotection against bleeds is maintained towards the end of a dosingperiod—the in vivo concentration of the antibody being lowest in thefinal days before the next dose is due. It may also assist in protectingareas of the body which are relatively poorly perfused by thecirculation—including the joints, which are a common site of problematicbleeding in haemophiliac patients.

Further aspects of the invention relate to pharmaceutical compositionscomprising the bispecific antigen-binding molecules and their use inmedicine including for the treatment of haemophilia A, as set out in theappended claims and described in the present disclosure.

Monospecific antibodies are also provided as aspects of the presentinvention. Thus, an anti-FIXa antibody may comprise two copies of afirst heavy-light chain pair as defined herein. An anti-FX antibody maycomprise two copies of a second heavy-light chain pair as definedherein.

Further aspects include nucleic acid molecules encoding sequences of theantibodies described herein, host cells containing such nucleic acids,and methods of producing the antibodies by culturing the host cells andexpressing and optionally isolating or purifying the antibodies. Theexpressed antibody is thereby obtained. VH and VL domains of antibodiesdescribed herein may similarly be produced and are aspects of thepresent invention. Suitable production methods of antibodies includelarge-scale expression from host cells (e.g, mammalian cells) in abioreactor by continuous or batch culture (e.g., fed batch culture).

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and embodiments of the invention will now be described in moredetail, with reference to the drawings, in which:

FIG. 1 illustrates the blood coagulation cascade [6].

FIG. 2 shows (A) co-factor action of FVIIIa interacting with FIXa andFX; (B) co-factor action of bispecific antibody interacting with FIXaand FX; and (C) bispecific antibody interacting with FIXa (9a) and FX(10) on the surface of a platelet. Bispecific antibody in thisembodiment is a four chain molecule having two disulphide-linked heavychains each comprising (N to C) domains VH1-CH1-CH2-CH3 and twoidentical light chains (common light chain) comprising (N to C) domainsVL-CL. In this illustration, binding site comprising VH1-VL binds to FXand binding site comprising VH2-VL binds to FXa.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 334) of factor IX, withresidue numbering for the mature protein. The signal peptide (straightunderlined) is cleaved after secretion. The propeptide (wave underlined)is cleaved on maturation. Mature factor IX contains a light chain(residues 1-145) and a heavy chain (residues 146-415). The activationpeptide (boxed) is cleaved on activation, generating activated factorIXa which contains a light chain (residues 1-145) and a heavy chain(residues 181-415, bold) joined by a disulphide bridge between Cys132and Cys289.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 335) of factor X, withresidue numbering. Residues 1-31 are a signal peptide (straightunderlined). Residues 32-40 are a propeptide (wave underlined). The FXlight chain is residues 41-179. The FX heavy chain is residues 183-488.The FXa heavy chain is residues 235-488 (bold).

FIG. 5 shows an embodiment of the invention: bispecific IgG with commonlight chain.

FIG. 6 summarises the process of optimising the anti-FIXa VH domainsequence N0436H and the anti-FX VH domain sequence T0200 for functionalcombination with each other and for pairing with the N0128L common VLdomain.

FIG. 7 illustrates (A) principles of in vitro assay for FVIII mimeticactivity of a bispecific molecule (FXase or tenase assay); and (B)example data from the assay showing positive result for FIXa-FXbispecific molecule compared with negative control.

FIG. 8 shows the results of screening bispecific antibodies havingvarious anti-FX arms in the FXase assay (standard reaction conditions).The bispecific antibody panel comprises a range of anti-FX VH testdomains, each in combination with the N0128H anti-FIX VH domain and0128L common VL domain.

FIG. 9 illustrates the B cell cluster identified for the lineage of theanti-FX T0200H domain.

FIG. 10 shows the results of screening optimised bispecific antibodiesin the FXase assay. (A) FXase activity for IgG4 bispecific antibodiescomprising named anti-FX VH domain combined with the N0128H, N1172H orN1280H anti-FIX VH domain and 0128L common light chain. OD at 405 nm at10 minutes (600s). (B) FXase activity for IgG4 bispecific antibodiescomprising named anti-FX VH domain combined with the N1280H anti-FIX VHdomain and N0128L common light chain.

FIG. 11 is an amino acid sequence alignment of a selection of anti-FX VHdomains from the T0200H B cell cluster. Germline sequence (SEQ ID NO:518) is shown for comparison. CDRs are boxed.

FIG. 12 identifies mutants of the T0201H VH domain in which the terminalfour residues of CDR3 were individually mutated to other amino acids.For example the T0590H VH domain is a Cys114Ala mutant of the T0201H VHdomain, i.e., in which the cysteine at IMGT position 114 is replaced byalanine.

FIG. 13 shows the amino acid sequence of VH domain T0201H, annotated in(A) to show FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and in (B) to showIMGT numbering.

FIG. 14 shows the amino acid sequence of VH domain N0128H, alignedagainst N192H and N1280H and annotated in (A) to show FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4, and in (B) to show IMGT numbering.

FIG. 15 shows the kinetics of activity of (A) IXAX-1280.0201.0128 and of(B) the comparator antibody AbE in the FXase assay. Antibody waspurified by Protein A and is composed of a homodimer/heterodimermixture.

FIG. 16 shows results of bispecific antibodies in Xase assay. “E” isAntibody E positive control. FXase activities are plotted as OD405 nm at10 minutes for samples normalised to 12.5 μg/mL (10.4 nM) finalconcentration. Bispecific antibodies are ranked in order of activity.

FIG. 17 shows the effect of bispecific antibody optimisation on FVIIImimetic activity (FXase). Results are shown for of bispecificantibodies, each having the named VH domain in the anti-FIXa arm andT0638H VH domain in the anti-FX arm, both paired with the 0128L commonlight chain VL domain. FVIII mimetic activity (FXase) was measured usingan in vitro chromogenic Factor X activation assay at 405 nm (Y-axis) asa function of time in seconds (X-axis). Optimised bispecific antibodiesare individually labelled. A human IgG4 isotype control demonstrates noFVIII mimetic activity. The key shows the name of the VH domain in theantibody to the right of its corresponding graph in order of maximalFXase activity, i.e., N1333H>N1280H>N1172H>N1091H>N0511H>N0436H>N0128H>control.

FIG. 18 shows aPTT assay results for IgG4 bispecific antibodiescomprising named anti-FX VH domain combined with the N0128H, N1172H orN1280H anti-FIX VH domain and 0128L common light chain.

FIG. 19 shows a dose response for IXAX-1280.0201.0128 bispecificantibody (purified on Protein A) in a one-stage activated aPTT clottingassay using FVIII-depleted human plasma.

FIG. 20 shows VH domain amino acid sequences of N0128H, N0436H, N0511H,N1091H, N1172H, N1280H, N1314H, N1327H and N1333H, annotated to identifytheir framework regions and CDRs.

FIG. 21 is an SPR sensorgram of bispecific antibody binding to antigen,where simultaneous binding to FIX and FX is demonstrated, compared withsensorgram of (A) isotype control antibody or (B) monospecific antibody.

FIG. 22 presents summary results of FXase assays for selected CDR1 andCDR2 sequence variants of T0681H. Except where indicated for AbEcontrols, samples were purified by Protein A chromatography and theirfinal concentrations normalised to 12.5 μg/mL (10.4 nM). 3 samples ofAbE positive control antibody are included: (i) protein A purified; 12.5μg/mL; (ii) protein A purified; 37.5 μg/mL; (iii) protein A purifiedfollowed by further purification by ion exchange chromatography; 37.5μg/mL. All data shown were sampled at the 10 minute time-point.

FIG. 23 summarises the effect of T0201H CDR1, CDR2 and CDR3 mutagenesison bispecific antibody FXase activity in vitro. Bispecific antibodyanti-FX T0201H variant VH domains were expressed with anti-FIX N01280Harm and N128_IgL common light chain in HEK cells, purified by Protein Achromatography and normalised by concentration to 0.15 mg/ml (125 nMfinal concentration). In vitro FXase assay was performed using 5 μl ofpurified material. Data shown are at 10 minute time point. Dotted linerepresents FXase activity of T0201H.

FIG. 24 presents summary results of aPTT clotting assays investigatingthe effect of sequence variation in the anti-FX T0201H VH domain onbispecific antibody activity. Clotting time (seconds) was recorded afterspiking FVIII deficient plasma with bispecific antibodies comprising thenamed anti-FX VH arm, anti-FIX N01280H arm and N128_IgL common lightchain. Bispecific antibodies were expressed in HEK cells, purified byProtein A chromatography and analysed at three different concentrations0.1 mg/ml, 0.3 mg/ml and 0.5 mg/ml. Dotted lines indicate the clottingtimes of FVIII deficient plasma spiked with a human IgG4 isotype controlor normal pooled human plasma spiked with PBS.

FIG. 25 identifies CDRs of VH domains which were progressively improvedfor FVIII mimetic activity during the mutagenesis process. Black shadingidentifies VH domains better than those in the same sub-table.

FIG. 26 shows the thrombin generation curve of normal pooled plasmausing factor IXa as a trigger in a thrombin generation assay (TGA). Thethrombogram depicts the generation of thrombin over time in sampleplasma, plotted as the concentration (nM) of thrombin generated duringclotting over time (minutes).

FIG. 27 describes the thrombin generation of FVIII deficient plasmaspiked with bispecific antibodies at final concentration of 133 nMcomprising CDR1, CDR2 and CDR3 single and combinatorial variants ofT0201H, compared with AbE, isotype control, normal plasma and emicizumabcalibrator. FIXa trigger at 0.3 nM. (A) 80 minute thrombogram. (B) 25minute thrombogram.

FIG. 28 shows thrombin Cmax (nM) and Tmax (min) from TGA carried out onFVIII deficient plasma spiked with bispecific antibodies comprisingCDR1, CDR2 and CDR3 single and combinatorial mutants of T0201H, comparedwith AbE, isotype control, normal plasma and emicizumab calibrator. Testantibodies were purified by Protein A chromatography only and thereforerepresent a mixture of heterodimer and homodimer. (A) Antibodyconcentration 133 nM. (B) Antibody concentration 80 nM.

FIG. 29 illustrates Cmax (nM) and Tmax (min) dose responses from TGAcarried out on FVIII deficient plasma spiked with (A) bispecificantibody IXAX-1280.0999.0128 and (B) emicizumab calibrator. Isotypecontrol (plus sign) used was a human IgG4 and was spiked into FVIIIdeficient plasma. Normal pooled plasma (cross sign) was spiked with PBS.

FIG. 30 shows dose response curves for Cmax of IXAX-1280.0999.0325 andAbE in TGA. Vertical dotted lines indicate final antibody concentrationcorresponding to 4.5 μg/ml (30 nM), 10 μg/ml (66.6 nM) and 45 μg/ml (300nM). Horizontal lines represent Cmax of normal pooled plasma collectedfrom healthy volunteers.

FIG. 31 shows thrombograms of (A) IXAX-1280.0999.0325 “BiAb_1” and (B)AbE, in human FVIII-depleted plasma, at antibody concentrations of 0.1nM, 1 nM, 10 nM, 100 nM and 300 nM. Thrombogram of a normal human plasmasample is shown in shadow.

FIG. 32 shows dose response curves for Tmax of IXAX-1280.0999.0325 andAbE in TGA. Vertical dotted lines indicate antibody concentrationcorresponding to 4.5 μg/ml (30 nM), 10 μg/ml (66.6 nM) and 45 μg/ml (300nM). Horizontal lines represent Tmax of normal pooled plasma collectedfrom healthy volunteers.

FIG. 33 shows thrombin generation abilities in terms of (A) Cmax and (B)Tmax of BiAb_1 (IXAX-1280.0999.0325), BiAb_2 (IXAX-1454.0999.0325),BiAb_3 (IXAX-1441.0999.0325) and BiAb_4 (IXAX-1442.0736.0325) comparedwith commercially available emicizumab calibrator in a TGA assay incommercially available human FVIII-depleted plasma.

FIG. 34 shows thrombin generation abilities in terms of (A) Cmax and (B)Tmax of BiAb_1 (IXAX-1280.0999.0325) compared with commerciallyavailable emicizumab calibrator in a TGA assay in human FVIII-depletedplasma.

FIG. 35 shows purification yield and % heterodimer for bispecificantibody IXAX-1172.0201.0128 expressed from 8 independent minipools ofstably transfected CHO cells.

FIG. 36 plots the correlation between bispecific antibody activity byFXase assay (activity at 10 min) and % heterodimer for bispecificantibody IXAX-1172.0201.0128 normalised to 0.3 mg/ml expressed from 8independent minipools. Pearson's correlation coefficient was calculatedas 0.9939.

FIG. 37 (A) shows separation of IXAX-1280.0999.0128 by ion exchangechromatography on a 1 ml CaptoSP ImpRes column and a linear NaClgradient up to 500 nM in 20 nM sodium phosphate, pH 6.0. Absorbance, mAU(milli absorbance unit); conductivity, mS/cm (milli Siemens percentimetre). Peak 1 is NINA-1280.0128 monospecific anti-FIX antibody.Peak 2 is IXAX-1280.0999.0128 bispecific antibody. Peak 3 isTINA-0999.0128 monospecific anti-FX antibody. (B) shows separation ofIXAX-0436.0202.0128 by ion exchange chromatography with stepwiseelution. Peak 1 is NINA-0436.0128 monospecific anti-FIX antibody. Peak 2is IXAX-0436.0202.0128 bispecific antibody. Peak 3 is TINA-0202.0128moospecific anti-FX antibody. (C) shows separation ofIXAX-1172.0201.0128 by ion exchange chromatography. Peak 1 isNINA-1172.0128 anti-FIX monospecific antibody. Peak 2 isIXAX-1172.0201.0128 bispecific antibody.

FIG. 38 shows cation exchange purification of purified FIX/FXheterodimers for each of bispecific antibodies (A) IXAX-1280.0999.0325(B) IXAX-1454.0999.0325 (C) IXAX-1441.0999.0325 and (D)IXAX-1442.0736.0325.

FIG. 39 shows dose response in the FXase assay with IXAX-1280.0999.0325and AbE. Dotted lines indicate antibody concentration corresponding to4.5 μg/ml, 10 μg/ml and 45 μg/ml.

FIG. 40 shows dose response in a chromogenic FVIII mimetic activityHyphen assay with IXAX-1280.0999.0325 and AbE.

FIG. 41 shows dose response in the aPTT assay with IXAX-1280.0999.0325and AbE. Vertical dotted lines represent 66.6 nM (10 μg/ml) and 300 nM(45 μg/ml) final antibody concentration; horizontal lines represent aPTTvalue of normal pooled plasma collected from healthy volunteers.

FIG. 42 presents results of aPTT clotting time assays investigating theeffect of bispecific antibodies IXAX-1280.0999.0325 (circle),IXAX-1441.0999.0325 (diamond) and AbE (cross) in inhibitor plasma. Doseresponses are shown for these antibodies in a one-stage aPTT clottingassay using plasma obtained from a patient with haemophilia Ademonstrating a specific inhibitor level of 70 BU to FVIII. Dottedhorizontal line indicates the clotting time of the inhibitor plasmaspiked with a human IgG4 isotype control.

FIG. 43 shows a thrombin peak height (Cmax) dose response for IgG4bispecific antibodies IXAX-1280.0999.0325, IXAX-1441.0999.0325 and AbE(all purified on Protein A, followed by cation exchange chromatography)in a thrombin generation assay with plasma obtained from a patient withhaemophilia A demonstrating a specific inhibitor level of 70 BU toFVIII. Bispecific antibody concentrations in nM are indicated for eachdilution.

FIG. 44 shows a dose response for IgG4 bispecific antibodies (A)IXAX-1280.0999.0325, (B) IXAX-1441.0999.0325 and (C) AbE (all purifiedon Protein A, followed by cation exchange chromatography) in a thrombingeneration assay with plasma obtained from a patient with haemophilia Ademonstrating a specific inhibitor level of 70 BU to FVIII. Bispecificantibody concentrations analysed are 100, 33.3, 11.1, 3.7 and 1.23 nM.Grey shaded area indicates thrombin generation of normal pooled plasma.TGA trigger is FIXa.

DETAILED DESCRIPTION

Blood Coagulation

The blood coagulation cascade is diagrammed in FIG. 1. Coagulation orclotting is one of the most important biological processes which stopsblood loss from a damaged vessel to allow the vessel to be repaired. Themechanism of coagulation involves activation, adhesion, and aggregationof platelets along with deposition and maturation of fibrin.Misregulation of coagulation can result in excessive bleeding(haemophilia) or obstructive clotting (thrombosis). Coagulation ishighly conserved in all mammals. It is controlled by a complex networkof coagulation factors. Coagulation is initiated when the endotheliumlining the blood vessel is damaged. The exposure of subendothelialtissue factor (TF) to plasma factor VII (FVII) leads to primaryhaemostasis (extrinsic pathway): a loose plug is formed at the site ofinjury. Activation of additional coagulation factors, especially factorIX (FIX) and factor VIII (FVIII), leads to secondary haemostasis(intrinsic pathway): fibrin strands are formed to strengthen the plug.Extrinsic and intrinsic pathways ultimately converge to a common point:the formation of the factor Xa/Va complex which together with calciumand bound on a phospholipid surface generate thrombin (factor IIa) fromprothrombin (factor II).

FVIII is cleaved by thrombin or factor Xa (FXa), and the resultantfactor Villa (FVIIIa) presents a heterotrimeric structure consisting ofthe A1 subunit, the A2 subunit, and the light chain. Upon activation andin the presence of calcium ions and a phospholipid surface (onplatelets), FVIIIa binds via its light chain and A2 subunit to FIXa andsimultaneously binds via its A1 subunit to FX, forming an activeintrinsic “tenase” or “Xase” complex in which the FVIIIa cofactor bringsFIXa and FX into proximity and also allosterically enhances thecatalytic rate constant of FIXa. See FIG. 2a . Factor X is activated bythe serine protease activity of FIXa, and the clotting cascadecontinues, culminating in the deposition of fibrin, the structuralpolymer of the blood clot.

Haemophilia arise through a deficiency in the Xase complex, due eitherto a lack of FVIII cofactor activity (haemophilia A) or a lack of FIXenzyme activity (haemophilia B).

Factor IX (FIX)

Factor IX is a serine protease which requires factor VIII as a cofactor.It circulates in blood as an inactive precursor, which is activatedthrough intrinsic or extrinsic pathway at the time of haemostaticchallenge, as discussed above.

Unless the context requires otherwise, factor IX referred to herein ishuman factor IX, and factor IXa is human factor IXa.

The amino acid sequence of human factor IX is shown in FIG. 3. Thefactor IX gene is approximately 34 kb in length and contains 8 exons.The transcript comprises a short 5′ untranslated region, an open readingframe plus stop codon and a 3′ untranslated region. The ORF encodes a461 amino acid pre-pro-protein in which the pre-sequence (signalpeptide) directs factor IX for secretion, the propeptide sequenceprovides a binding domain for a vitamin K dependent carboxylase, whichcarboxylates certain glutamic acid residues in the adjacent GLA domain,and the remainder represents the factor IX zymogen, which enters intocirculation after removal of the pre- and pro-sequences. The mature 415residue FIX protein contains, from N to C terminus: a GLA domain inwhich 12 glutamic acid residues are post-translationally γ-carboxylated,two epidermal growth factor (EGF)-like domains, an activation peptidesequence and a catalytic serine protease domain. FIX is activated byeither activated factor XI generated through the intrinsic pathway, orby the TF/FVIIa complex of the extrinsic pathway. Either way, activationinvolves cleavage of the peptide bond following R145 (α-cleavage) and ofthe peptide bond following R180 (β-cleavage), releasing an activationpeptide corresponding to the intervening sequence, and therebygenerating the activated FIXa molecule, which has an N terminal lightchain (GLA-EGF-EGF) and a C terminal heavy chain (catalytic domain)joined by a disulphide bridge between C132 of the light chain and C289of the heavy chain. Residue numbering refers to amino acids in themature FIX polypeptide sequence. On the phospholipid surface where theXase complex forms, it is the GLA domain of FIXa which associates withthe phospholipid, while the catalytic domain stands high (>70 Å) abovethe phospholipid surface and is modulated by the A2 domain of FVIIIa [7,8].

The molecular basis of haemophilia B—deficiency in FIXa activity—isdiverse, including a variety of point mutations, nonsense mutations,mRNA splice site mutations, deletions, insertions, or mis-sensemutations at activation cleavage sites [9].

The catalytic (protease) domain of activated FIX (FIXa) is involved inbinding to FVIIIa. Residue E245 in this domain binds calcium ions, andmutations at this position may reduce binding to FVIII and lead tohaemophilia B, for example the substitution E245V. Mutations within theFIX helix formed by residues 330-338 are also linked with reducedbinding to FVIII and consequently to haemophilia B.

Non-pathogenic mutations in factor IX have also been reported, includingsingle nucleotide polymorphisms (SNPs) and length polymorphisms—reviewedin [9]. These include the MnII SNP in exon 6, resulting in T/Asubstitution at residue 148 (Malmo polymorphism), which is relativelycommon among white and black American populations [9].

Factor X (FX)

Unless the context requires otherwise, factor X referred to herein ishuman factor X, and factor Xa is human factor Xa. The amino acidsequence of human FX is shown in FIG. 4.

FX is also known as Stuart-Prower factor. It is a serine endopeptidase.FX can be activated, by hydrolysis, into factor Xa by either factor IX(together with its cofactor, factor FVIII, as described above) or factorVII (with its cofactor, tissue factor). FX acts by cleaving prothrombinin two places—at an Arg-Thr bond and then at an Arg-Ile bond, to yieldthe active thrombin.

Antigen-Binding

A desirable feature of the bispecific antigen-binding molecule is thatit binds FIXa and FX in a manner that allows the bound FIXa to activatethe bound FX.

To bring FIXa and FX together and thereby promote the activation of FXby FIXa, the bispecific antigen-binding molecule may bind these twocofactors simultaneously. Binding may occur sequentially, e.g., aninitial binary complex may form between a first binding arm and itscognate antigen, followed by binding of the second binding arm to itscognate antigen. In principle these two binding events may occur ineither sequence, i.e., FIXa followed by FX, or FX followed by FIXa. Themolecular choreography is influenced by the relative affinities of thetwo binding sites for their respective antigens. In a population ofbispecific antigen-binding molecules, FIXa and FX, a number of differentcomplexes are expected to exist in parallel. Thus the pool will comprisefree antigen-binding molecule, free FIXa, free FX, FIXa complexed withantigen-binding molecule, FX complexed with antigen-binding molecule,and a tertiary complex of FIX, FX and antigen-binding molecule, witheach of these species being present in different proportions accordingto the relative on-rates and off-rates of the individual interactions.

It may be preferable for a bispecific antigen-binding molecule to have ahigher affinity for FIXa than for FX. Such a bispecific molecule wouldbe envisaged to form an initial complex with FIXa, which in turn wouldbind and activate FX. The relatively low affinity for FX reduces theproportion of FX that is bound in incomplete antibody-antigen complexes(i.e., without FIXa). A potential advantage of this is that it allows agreater proportion of FX to remain free to engage with any FVIII thatmay be present in a patient's blood. Haemophilia A encompasses a rangeof deficiencies in FVIII, ranging from mild deficiency to total absenceof functional FVIII. For those patients who retain some functionalFVIII, it may be desirable to retain this natural activity as far aspossible. Thus, it may be desirable to provide a bispecificantigen-binding molecule in which the FX binding arm does not competewith FVIII for binding to FX.

Preferably the FX binding arm has a higher affinity for FX than for FXa.A low affinity for FXa promotes release of the activated product,completing the role of the FVIII-mimetic molecule in the coagulationcascade and freeing the FX binding site for re-use. In variousembodiments, a bispecific described herein (e.g., antibodyIXAX-1280.0999.0325 or antibody IXAX-1441.0999.0325), the FX binding armof such a bispecific (e.g., binding arm comprising T0999H VH domain), oran anti-FX monospecific antibody comprising a homodimer of two sucharms, has at least 2-fold higher, at least 3-fold higher, at least4-fold higher, at least 5-fold higher, at least 10-fold higher, at least100-fold higher affinity for FX than for FXa, e.g., at least 1000-foldhigher affinity for FX than for FXa, and optionally does not showsignificant binding to FXa, e.g., as measured by ELISA. For example, invarious embodiments the bispecific, FX binding arm or anti-FXmonospecific antibody (e.g., TINA-0999.0325) does not bind human FXa asdetermined by ELISA and with reference to a negative control IgG. As analternative to ELISA, affinity may be measured by SPR and the affinityfor FX compared with affinity for FXa.

FIXa Binding

The FIXa binding arm of a bispecific antigen-binding molecule may bindthe light chain and/or the heavy chain of FIXa. Initial studiesindicated that FIXa binding arms of the N128 lineage described in theExamples do not bind the FIXa light chain in isolation (in the absenceof the heavy chain).

A bispecific antigen-binding molecule of the present invention (or FIXabinding polypeptide arm thereof) may thus be one which binds a FIXamolecule comprising a heavy chain and a light chain, and which does notbind the FIX light chain in the absence of the heavy chain. Optionally,the FIXa binding arm recognises an epitope formed by, or stabilised by,the combination of the FIXa heavy and light chains. It may for examplemake contact only with the light chain in the FIXa molecule, binding anepitope that is exposed or stabilised only when the light chain ispresent in combination with the heavy chain in the FIXa molecule.Alternatively, it may contact an epitope comprising one or more residuesfrom both the light chain and the heavy chain, or comprising residues ofthe heavy chain alone.

An antigen-binding molecule according to the present invention, or aFIXa-binding polypeptide arm thereof, may bind the EC domain of humanFIXa with an affinity (measured as KD) of 10 mM or less, preferably 5 mMor less, more preferably 1 mM or less. For example, KD may be between 1nM and 3 μM.

The KD for binding human FIXa may be between 0.1 μM and 1 μM, e.g.,between 0.15 and 0.3 μM. The KD may be 0.6 μM or less, 0.5 μM or less,0.4 μM or less, 0.3 μM or less, 0.25 μM or less, or 2 μM or less. The KDmay be at least 0.1 μM, for example at least 0.2 μM. It may be 0.1μM-0.5 μM.

The KD may be between 10 and 100 nM, e.g., between 25 and 75 nM.

The KD may be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less,or 1 nM or less. The KD may be 0.9 nM or less, 0.8 nM or less, 0.7 nM orless, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, or 0.1 nM or less. The KD may be at least 0.001 nM, forexample at least 0.01 nM or at least 0.1 nM. The KD may be between0.1-10 nM.

An antigen-binding molecule according to the present invention, or aFIXa-binding polypeptide arm thereof, may bind human FIX with anaffinity (measured as KD) between 0.1 μM and 1 μM, e.g., between 0.15and 0.3 μM. The KD may be 0.6 μM or less, 0.5 μM or less, 0.4 μM orless, 0.3 μM or less, 0.25 μM or less, or 2 μM or less. The KD may be atleast 0.1 μM, for example at least 0.2 μM.

The KD of interaction with FIX may be comparable to the KD ofinteraction with FIXa, e.g., there may be difference of less than 25%,optionally less than 10%, in the FIXa-binding arm's affinity for FIXcompared with the affinity for FIXa. There may be no statisticallysignificant difference in KD of interaction with FIX compared with FIXa.

As described elsewhere herein, affinity may be determined using surfaceplasmon resonance (SPR), e.g., with the binding arm coupled to a solidsurface, optionally as a dimer (e.g., as monospecific IgG), with theantigen in solution as analyte, at 25° C.

FX Binding

An antigen-binding molecule according to the present invention, or aFX-binding polypeptide arm thereof, may bind the EC domain of human FXwith a KD of 10 mM or less, preferably 5 mM or less, more preferably 1mM or less. For example, KD may be between 5 μM and 1 nM, e.g., between5 μM and 10 nM.

The KD may be between 0.1 μM and 2 μM, e.g., between 0.1 μM and 1 μM,e.g., between 0.15 and 0.3 μM. The KD may be 0.6 μM or less, 0.5 μM orless, 0.4 μM or less, 0.3 μM or less, or 0.25 μM or less. The KD may beat least 0.1 μM.

The KD may be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less,or 1 nM or less. The KD may be 0.9 nM or less, 0.8 nM or less, 0.7 nM orless, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, or 0.1 nM or less. The KD may be at least 0.001 nM, forexample at least 0.01 nM or at least 0.1 nM. For example, the KD may bebetween 1-100 nM. KD may be between 1-10 nM.

As described elsewhere herein, affinity may be determined using surfaceplasmon resonance (SPR), e.g., with the binding arm coupled to a solidsurface, optionally as a dimer (e.g., as monospecific IgG), with theantigen in solution as analyte, at 25° C.

Measurement of Antigen-Binding Affinity

The affinity of an antigen-binding molecule for binding FIX, FIXa, FXand FXa may be quantified in terms of the equilibrium dissociationconstant KD, the ratio Ka/Kd of the association or on-rate (Ka) and thedissociation or off-rate (kd) of the binding interaction. KD, Ka and Kdfor antigen binding can be measured using surface plasmon resonance(SPR). Example SPR procedure and conditions are set out in Example 10.

Quantification of affinity may be performed using SPR with theantigen-binding polypeptide arm in monovalent form, e.g., antibody Fabor Fv comprising the antigen binding site, or heterodimericimmunoglobulin (e.g., IgG) having a single antigen-binding arm for theantigen in question. Alternatively, it may be convenient to determineaffinity for the antigen-binding polypeptide arm in bivalent form, forexample IgG comprising homodimeric antigen-binding arms. SPR maycomprise coating dimers of the antigen-binding polypeptide arm on to abiosensor chip (directly or indirectly), exposing the antigen-bindingpolypeptide arms to antigen in buffered solution at a range ofconcentrations, detecting binding, and calculating the equilibriumdissociation constant KD for the binding interaction. SPR may beperformed at 25° C. A suitable buffered solution is 150 mM NaCl, 0.05%detergent (e.g., P20) and 3 mM EDTA, pH 7.6. HBS-P 1× (10 mM HEPES pH7.4, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20 pH 7.6) with 2.5 mMCaCl₂. is an example buffer. The binding data can be fitted to a 1:1model using standard algorithms, which may be inherent to the instrumentused. A variety of SPR instruments are known, such as Biacore™, ProteOnXPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc).

Cross-Reactivity

Regulatory bodies may require candidate therapeutic molecules to havedemonstrated therapeutic efficacy in laboratory animals before theyadvance to human clinical trials. An example of an acquired haemophiliaA animal model is a cynomolgus monkey that is rendered deficient inblood clotting through administration of a FVIII-neutralising antibodyor a small molecule inhibitor against FVIII, thereby replicating thephenotype of a human haemophilia A patient. To enable testing ofbispecific antigen-binding molecules in animal models, it is desirablefor the binding site of each arm to be cross-reactive with thecorresponding antigen from one or more non-human mammals. Thus, the FIXabinding site of the antigen-binding molecule may bind murine (e.g.,mouse or rat), rabbit or non-human primate (e.g., cynomolgus monkey)FIXa as well as human FIXa, and the FX binding site may bind murine(e.g., mouse or rat), rabbit or non-human primate (e.g., cynomolgusmonkey) FXa as well as human FXa.

One way to quantify the extent of species cross-reactivity of anantigen-binding molecule (or, more precisely, of its antigen bindingsite) is as the fold-difference in its affinity for antigen or onespecies compared with antigen of another species, e.g., fold differencein affinity for human antigen vs cynomolgus antigen. Affinity may bequantified as KD, referring to the equilibrium dissociation constant ofthe binding of the antigen to the antigen-binding molecule. KD may bedetermined by SPR as described elsewhere herein.

A species cross-reactive binding molecule may have a fold-difference inaffinity for binding human and non-human antigen that is 30-fold orless, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or lessor 5-fold or less. To put it another way, the KD of binding theextracellular domain of the human antigen may be within 30-fold,25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the KD of binding theextracellular domain of the non-human antigen.

Preferably, the binding affinities of human and non-human antigen arewithin a range of 10-fold or less, more preferably within 5-fold orwithin 2-fold. KD for binding non-human FIXa, e.g., as determined bysurface plasmon resonance, may be up to 10-fold (preferably up to 5-foldor up to 2-fold) greater or up to 10-fold lower (preferably up to 5-foldor up to 2-fold lower) than the Kd for binding human FIXa. Similarly, KDfor binding non-human FX, e.g., as determined by SPR, may be up to10-fold (preferably up to 5-fold or up to 2-fold) greater or up to10-fold (preferably up to 5-fold or up to 2-fold) lower than the Kd forbinding human FX. Methods of determining affinity are describedelsewhere herein.

Binding molecules can also be considered species cross-reactive if theKD for binding antigen of both species meets a threshold value, e.g., ifthe KD of binding human antigen and the KD of binding non-human antigenare both 10 mM or less, preferably 5 mM or less, more preferably 1 mM orless. The KD may be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nMor less. The KD may be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less,0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nMor less, or 0.1 nM or less.

While species cross-reactivity for binding antigen of different speciesmay be advantageous, selectivity of the FIXa binding arm and the FXbinding arm for their respective antigens is nevertheless desirable toavoid unwanted side effects. Thus, within the body, FIX/FIXa and FX/FXaare preferably the only antigens bound by the antigen-binding molecule.

Enhancement of FIXa-Mediated Activation of FX

The ability of a bispecific antigen-binding molecule to enhance theFIXa-mediated activation of FX to FXa may be determined in assays invitro or in vivo.

A suitable in vitro assay is the FX activation assay exemplified inExample 3 and Example 7 and illustrated in FIG. 7. The assay comprises

-   -   (i) contacting the bispecific antigen-binding molecule with FIXa        and FX under conditions suitable for formation of FXa (e.g., in        the presence of phospholipid, in buffered solution at 37° C.)    -   (ii) adding substrate that is cleavable by FXa to generate a        detectable product, and    -   (iii) detecting, and optionally quantifying, the presence of the        detectable product.

A detailed protocol is set out in Example 7.

The level of product may be compared with a control assay in whichFIXa-FX bispecific antigen-binding molecule is absent from the reactionmixture. Significant difference in product level in the assay with thebispecific compared with control indicates that the bispecific is ableto enhance FIXa-mediated activation of FX. FVIII may be included as apositive control.

The level of product may be compared with an assay in which the FIXa-FXbispecific antigen-binding molecule is emicizumab. A bispecificaccording to the present invention may enhance the FIXa-mediatedactivation of FX to FXa to the same or similar extent (e.g., within 10%difference or within 5% difference) as emicizumab, or to a greaterextent (e.g., more than 10% more activation of FX to FXa than isachieved with emicizumab as measured by the level of detectableproduct). Preferably the bispecific antibody enhances the FIXa-mediatedactivation of FX to FXa to at least the same extent as emicizumab. Theassay is typically performed at physiological temperature of 37 degreesC. Suitable concentrations of bispecific for use in the assay areindicated in the Examples herein, e.g., 12.5 μg/ml (10.4 nM) or 125 nM.

Another suitable assay is to measure the activated partialthromboplastin time (aPTT) in FVIII-deficient plasma, which may beperformed in the presence or the absence of inhibitors and can be usedto compare the activity of bispecific molecules with recombinant humanFVIII. This assay is exemplified in Example 8. aPTT is an end pointassay which provides a global overview of blood clot formation andprovides coagulation time as the assay read-out. FVIII-deficient plasmawould typically have a coagulation time of around 80-90 seconds in theaPTT assay. Bispecific antigen binding molecules of the presentinvention are effective to reduce the coagulation time in an aPTT assay(compared with a negative control). The coagulation time of humanFVIII-deficient in an aPTT assay with a bispecific antigen bindingmolecule according to the present invention may for example be the sameas or less than that of the coagulation time with recombinant humanFVIIIa. Physiological clotting time for normal (FVIII+) human plasma istypically <40 seconds, e.g., in the range of 37-34 s. Similar values areachievable with FVIII-deficient plasma upon provision of activatedFVIIIa, which provides a convenient way of standardising the assaythrough calibration of the apparatus/measurement against referencevalues. Alternatively, coagulation time of normal (FVIII+) human plasmamay be used for reference, the aPTT assay being begun by induction ofcoagulation through the addition of calcium. The assay is typicallyperformed at physiological temperature of 37 degrees C. Suitableconcentrations of bispecific for use in the assay are indicated in theExamples herein, and include 0.1 mg/ml (44 nM), 0.3 mg/ml (133 nM) and0.5 mg/ml (222 nM).

A bispecific antigen-binding molecule of the present invention may givea coagulation time in the aPTT assay of within 10 seconds of that ofFVIIIa (i.e., up to 10 seconds more than or up to 10 seconds less thanthe coagulation time of the aPTT assay with FVIIIa). Preferably, thecoagulation time in the aPTT assay with a bispecific antigen bindingmolecule of the invention is less than that with FVIIIa. The bispecificantigen-binding molecule may reduce the coagulation time to less than 40seconds, less than 35 seconds, or less than 30 seconds. The coagulationtime may be between 20 and 40 seconds, e.g., between 20 and 30 seconds.Preferably the coagulation time is 22-28 seconds, e.g., 24-26 seconds.

Another measure of function is the rate at which thrombin is generatedin FVIII-deficient blood plasma in the presence of the bispecificantigen-binding molecule. Activity of a bispecific antibody may bemeasured in a thrombin generation assay (TGA) [10]. A number of thrombingeneration assays have been described, as recently reviewed [11].Essentially, a TGA comprises measuring the conversion (activation) ofprothrombin to thrombin over time following addition of a test molecule(here, the candidate bispecific antibody), where thrombin is detectedvia its cleavage of a substrate to form a detectable product.

With reference to FIG. 1, it will be remembered that the extrinsictissue factor (TF) pathway exists to initiate the coagulation cascade.Cells expressing TF normally reside outside of the vasculature, and upontissue damage such TF-bearing cells come into contact with circulatingplatelets. TF acts as a co-factor to facilitate the activation of smallamounts of factors IX and X by factor VIIa. Activated factor Xa andfactor V form a prothrombinase complex on TF-bearing cells, generating alimited amount of thrombin. The newly generated thrombin activatesplatelets which have accumulated at the site of injury and factor XIwhich is present on the platelets. Platelet bound FXIa is required toensure further activation of FIXa. Given the TGA is an ex vivo assay,TF-bearing cells are absent and a coagulation trigger must be suppliedto initiate the cascade. Commercial assays typically use a recombinantTF/phospholipid mixture to initiate coagulation [11]. The TGA methodexemplified herein (see Example 13) uses a factor IXa/phospholipidmixture as the trigger, although other upstream activators such as FXIacould be used.

To perform the TGA, FVIII-deficient plasma is contacted with (i) thetrigger reagent, (ii) a substrate convertable by thrombin to adetectable product, e.g., a fluorogenic or chromogenic substrate whichproduces a visually detectable product on cleavage by thrombin, and(iii) the test molecule (e.g., bispecific antibody), to createconditions under which the presence of FVIII-mimetic activity wouldresult in thrombin generation and hence a signal from the detectableproduct. Typically, the plasma will lack free metal ions such ascalcium, which are required in the blood clotting cascade (FIG. 1). Ca²⁺ions may be supplied (e.g., as CaCl₂ in solution) to initiate the assay,e.g., it may be contained within the substrate solution. Followinginitiation, generation of the detectable product (representinggeneration of thrombin) is monitored (preferably continuously, or atfrequent intervals, e.g., about every 20 seconds) over time, e.g., bydetecting fluorescence or colour. A plate reader may be used, e.g., tomonitor conversion of a fluorogenic substrate into a fluorophore. TGA isperformed at physiological temperature of 37 degrees C.

Fluorescence may be converted to thrombin concentration by calibratingagainst known concentrations of thrombin added to control plasma. Athrombogram may then be generated (FIG. 26, FIG. 27).

Preferably, bispecific antibodies (or other test molecules) are suitablypurified for use in the TGA (e.g., by protein A chromatography and ionexchange chromatography or hydrophobic interaction chromatography),e.g., to provide the bispecific in a composition of at least 95bispecific heterodimer (i.e., no more than 5% homodimeric or otherantibody contaminants should be present). Preferably the test moleculeis provided as close to 100% purity as possible. It may be about 98, 99%or 100% pure bispecific.

Approximate reference ranges for plasma from healthy individuals in afluorogenic TGA are Cmax 200 to 450 nM and Tmax 5 to 8 minutes [11].Activity in a TGA can also be compared against published representativethrombin generation curves for plasma from healthy individuals, patientswith severe FVIII deficiency and patients with severe FVIII deficiencyafter FVIII infusion [12]. For standardisation, performance in the TGAmay also be compared against a calibrator which represents a positivecontrol molecule at known concentration. A dilution series of the testbispecific may be compared against the calibrator at a series of knownfixed concentrations. A suitable calibrator is an emicizumab calibrator.Emicizumab calibrator is available commercially, prepared from FVIIIimmunodepleted citrated human plasma spiked with 100 μg/mL emicizumab(Hemlibra®) and further comprising buffer and stabilisers. It issupplied in lyophilised form and is reconstituted in water before use inthe TGA. The exact concentration of emicizumab in the calibrator phialis known, so the activity of a test bispecific molecule in the assay canbe compared against the activity of the calibrator after normalising forconcentration. As an alternative control for comparison of a bispecificantibody against emicizumab, performance of the test bispecific antibodyin the TGA may be compared against performance of a control bispecificantibody having the amino acid sequence of emicizumab, wherein the testbispecific antibody and the bispecific antibody having the amino acidsequence of emicizumab are tested under identical conditions in the TGA.

The TGA may be used to characterise six aspects of thrombin generation:lag time (lag), time to peak (Tmax), maximal peak height (Cmax),endogenous thrombin potential (ETP), velocity index (VI) and the “tailstart” or return to baseline. The lag time represents the initiationphase before the thrombin peak begins to be generated, where addition ofa trigger results in the activation of the coagulation cascade. Onceinitiated, large amounts of thrombin are quickly generated during thepropagation phase. The time to peak represents the time taken (Tmax) toreach maximal thrombin peak height (Cmax), the ETP represents the totalamount of thrombin generated and the velocity index characterises theslope between the lag time and the time to peak. The return to baseline(tail start) reflects the inhibition (by activated protein C) ofthrombin formation and the inactivation (by antithrombin) of thrombinalready formed. The Cmax and/or Tmax is typically the key measure usedto represent activity in the TGA. References values in the TGA (e.g.,Cmax, Tmax, lagtime etc.) may be determined for the bispecific at afixed concentration, e.g, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM or 300 nM.Parameters may be measured a series of concentrations, e.g., at 1 nM, 3nM, 10 nM, 30 nM, 100 nM and 300 nM and/or other concentrations toobtain a complete dose response curve, allowing EC50 values to then bedetermined. The dose response curve can be fitted using a non-linearlog(antibody) vs response variable slope model (e.g., variable slope 4parameter logistic regression model, which may be performed usingGraphPad Prism v8.0.0). EC50 is the concentration of test molecule(e.g., antibody) at which half-maximal effect is reached (half waybetween baseline and maximal value of the measured parameter). EC50 canbe determined from the dose response curve. Worked examples with EC50data are presented in Example 14 herein.

In one embodiment, the TGA comprises:

(a) contacting FVIII-deficient plasma lacking free calcium ions with

-   -   (i) a trigger reagent comprising a factor IXa/phospholipid        mixture,    -   (ii) a solution comprising a fluorogenic substrate (e.g., 2 nM        ZGGR-AMC fluorogenic substrate) which produces a visually        detectable fluorophore on cleavage by thrombin, and calcium ions        (e.g., 100 mM CaCl2)), (for example, the FluCa reagent from        Stago), and    -   (iii) the bispecific antibody (e.g., at a purity of 95%-100%,        e.g., 99%-100%),        (b) incubating the plasma at 37 degrees C. under conditions in        which the presence of FVIII-mimetic activity would result in        thrombin generation and hence a signal from the fluorophore,        (c) detecting fluorescence over time to monitor conversion of        the fluorogenic substrate to fluorophore,        (d) calibrating detected fluorescence against fluorescence from        solutions of thrombin at predetermined concentrations, and        (e) determining one or more parameters of a thrombogram, wherein        said parameters are:    -   maximal thrombin peak height (Cmax) that is reached,    -   time taken (Tmax) to reach maximal thrombin peak height, and/or    -   length of the initiation phase before the thrombin peak begins        to be generated (lag time).

Said one or more parameters are determined at a series of concentrationsof the bispecific antibody to obtain a complete dose response curveincluding baseline and top plateau (maximal value) of response. A doseresponse curves may be fitted to the data points using a non-linear log[antibody] vs response parameter variable slope model (4 parameterlogistic regression model). EC50 is determined from said dose responsecurve. Said one or more parameters, or EC50 for said one or moreparameters, may be compared between the test bispecific antibody andemicizumab (e.g., emicizumab calibrator, as available from EnzymeResearch Laboratories).

A bispecific antibody according to the present invention preferablyexhibits a potency that is similar to or greater than that of emicizumabin a fluorimetric TGA. Higher potency may be represented by lower EC50for one or more parameters in said assay, e.g., Cmax, Tmax or lagtime.As demonstrated in the Examples herein, embodiments of the presentinvention consistently demonstrated greater potency than emicizumab atlower concentrations. See, for example, the results presented in FIG.29, FIG. 30, FIG. 31, FIG. 33 and FIG. 34.

The maximal response (e.g., highest Cmax, lowest Tmax, shortest lagtime,etc) in the fluorimetric TGA is also noteworthy. Maximal response is thelevel at which the measured parameter (e.g., Cmax) plateaus withincreasing antibody concentration, and represents the maximum achievablelevel (e.g., the maximal Cmax). An excessive maximal response may beassociated with increased risks of overdosing the bispecific molecule,including risk of consumption coagulopathy or disseminated intravascularcoagulation (DIC) which is characterised by abnormally increasedactivation of procoagulant pathways. Hypercoagulability may compromisepatient safety through coagulopathy events such as arterial/venousthrombosis, embolism and thrombotic microangiopathy, and would thusnarrow the therapeutic window, i.e., the range of dose or plasmaconcentration at which a beneficial effect is achieved withoutunacceptable side effects or risk of adverse events.

Since emicizumab has received regulatory approval based on a safetyprofile deemed acceptable in human clinical trials, the maximal responseof emicizumab in the TGA represent established safe limits. Optionally,bispecifics of the present invention have a maximal Cmax and/or maximalTmax response in the TGA which is not more than 20% (e.g., not more than15 or not more than 10%) different from that of emicizumab. Thesereference values may be determined using an emicizumab calibrator or asequence identical analogue of emicizumab.

Bispecifics of the present invention may demonstrate maximal responsesin the TGA as follows:

-   -   Cmax in the TGA not exceeding 500 nM. Optionally the maximal        response for Cmax does not exceed 450 nM, e.g., does not exceed        400 nM. Maximal response for Cmax may be between 200 and 450 nM,        e.g., between 250 and 350 nM; and/or    -   Tmax in the TGA not lower than a maximal response of 1 minute.        Optionally the maximal response for Tmax is not less than 5        minutes, not less than 4 minutes, not less than 3 minutes or not        less than 2 minutes. Maximal response for Tmax may be between 2        and 10 minutes, e.g., between 2 and 8 minutes or between 5 and 8        minutes.

A bispecific antigen-binding molecule according to the present inventionmay have a Cmax in the range of 100 to 450 nM (e.g., 200 to 450 nM) asdetermined by fluorimetric TGA, e.g., wherein the bispecific antibody isat a concentration of 100 nM or 300 nM in said assay. The Cmax ispreferably at least 200 nM, more preferably at least 250 nM or at least300 nM. The Cmax of the bispecific may be the same or similar to (e.g.,within 10% difference from) the Cmax of emicizumab, or it may be greaterthan that of emicizumab. The bispecific may have a Cmax EC50 in saidassay that is within 10% of the Cmax EC50 of emicizumab, or that islower. Where the EC50 is lower than that of emicizumab, there may be atleast a 2-fold, at least a 3-fold, at least a 4-fold or at least a5-fold difference in Cmax EC50 in the TGA between the bispecific of thepresent invention and emicizumab. Optionally the Cmax EC50 in the TGAmay be up to 10-fold, up to 15-fold or up to 20-fold different. EC50 ofthe Cmax for the bispecific antigen-binding molecule in the fluorimetricTGA may be less than 50 nM, e.g., between 1 nM and 50 nM, between 5 nMand 20 nM, or between 5 nM and 10 nM.

A bispecific antigen-binding molecule according to the present inventionmay have a Tmax of 8 minutes or under, e.g., in the range of 4 to 8minutes, as determined by fluorimetric TGA, e.g., wherein the bispecificantibody is at a concentration of 100 nM or 300 nM in said assay. TheTmax of the bispecific may be the same or similar to (e.g., within 10%difference from) the Tmax of emicizumab, or it may be less than that ofemicizumab. The bispecific may have a Tmax EC50 in said assay that iswithin 10% of the Tmax EC50 of emicizumab, or that is lower.

EC50 of the Tmax for the bispecific antigen-binding molecule in thefluorimetric TGA may be less than 5 nM, e.g., less than 3 nM or lessthan 2 nM. It may be between 1 nM and 5 nM, e.g., between 1 nM and 2 nM.

A bispecific antigen-binding molecule according to the present inventionmay have a lag time of 2-6 minutes as determined by fluorometric TGA,e.g., wherein the bispecific antibody is at a concentration of 100 nM or300 nM in said assay. The lagtime of the bispecific may be the same orsimilar to (e.g., within 10% difference from) the lagtime of emicizumab,or it may be lower than that of emicizumab. The bispecific may have alagtime EC50 in said assay that is within 10% of the lagtime EC50 ofemicizumab, or that is lower.

Bispecific Antigen-Binding Molecules

The bispecific antigen-binding molecule comprises a FIXa bindingpolypeptide arm and a FX binding polypeptide arm. It may be amulti-chain or single-chain polypeptide molecule. While the FIXa bindingpolypeptide arm and the FX binding polypeptide arm represent differentmoieties of the bispecific molecule, one polypeptide can optionally formall or part of both the FIXa binding arm and the FX binding arm.

A polypeptide binding arm is the region of the bispecific molecule thatcomprises the binding site for one of the antigens (FIXa or FX). One orboth antigen-binding sites of a bispecific molecule can be provided by aset of complementarity determining regions (or peptide loops) in apolypeptide arm, wherein the polypeptide arm is any suitable scaffoldpolypeptide whether that of an antibody (e.g., an antibody Fv region) ora non-antibody molecule. A binding arm may comprise one or more than one(e.g., two) polypeptides or parts (e.g., domains) thereof.

The invention is described in detail herein with reference to bispecificantibodies, wherein at least one of the antigen binding polypeptide armsis provided by a set of CDRs in an antibody VH and/or VL domain,optionally an Fv region.

Antibodies are immunoglobulins or molecules comprising immunoglobulindomains. Antibodies may be IgG, IgM, IgA, IgD or IgE molecules ormolecules including antigen-specific antibody fragments thereof. Theterm “antibody” covers any polypeptide or protein comprising an antibodyantigen-binding site. An antibody antigen-binding site (paratope) is thepart of an antibody that binds to and is complementary to the epitope ofits target antigen. The term “epitope” refers to a region of an antigenthat is bound by an antibody. Epitopes may be defined as structural orfunctional. Functional epitopes are generally a subset of the structuralepitopes and have those residues that directly contribute to theaffinity of the interaction. Epitopes may also be conformational, thatis, composed of non-linear amino acids. In certain embodiments, epitopesmay include determinants that are chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl groups, orsulphonyl groups, and, in certain embodiments, may have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics.

An antibody antigen-binding site is provided by a set of complementaritydetermining regions (CDRs) in an antibody VH and/or VL domain, and iscapable of binding the antigen. In an example, the antibody binding siteis provided by a single variable domain, e.g., a heavy chain variabledomain (VH domain) or a light chain variable domain (VL domain). Inanother example, the binding site is provided by a VH/VL pair (an Fv) ortwo or more such pairs.

The antibody variable domains are the portions of the light and heavychains of antibodies that include amino acid sequences ofcomplementarity determining regions (CDRs; ie., CDR1, CDR2, and CDR3),and framework regions (FRs). Thus, within each of the VH and VL domainsare CDRs and FRs. A VH domain comprises a set of HCDRs, and a VL domaincomprises a set of LCDRs. VH refers to the variable domain of the heavychain. VL refers to the variable domain of the light chain. Each VH andVL is typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. Amino acid positions assigned to CDRs and FRsmay be defined according to IMGT nomenclature. An antibody may comprisean antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and aframework. It may alternatively or also comprise an antibody VL domaincomprising a VL CDR1, CDR2 and CDR3 and a framework. Example sequencesof antibody VH and VL domains and CDRs form part of the presentdisclosure. The CDRs are defined according to the IMGT system [13]. AllVH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs andsets of LCDRs disclosed herein represent aspects and embodiments of theinvention. As described herein, a “set of CDRs” comprises CDR1, CDR2 andCDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a setof LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a“set of CDRs” includes HCDRs and LCDRs.

An antibody may comprise one or more CDRs, e.g. a set of CDRs, within anantibody framework. The framework regions may be of human germline genesegment sequences. Thus, the antibody may be a human antibody having aVH domain comprising a set of HCDRs in a human germline framework.Normally the antibody also has a VL domain comprising a set of LCDRs,e.g. in a human germline framework. An antibody “gene segment”, e.g., aVH gene segment, D gene segment, or JH gene segment refers tooligonucleotide having a nucleic acid sequence from which that portionof an antibody is derived, e.g., a VH gene segment is an oligonucleotidecomprising a nucleic acid sequence that corresponds to a polypeptide VHdomain from FR1 to part of CDR3. Human v, d and j gene segmentsrecombine to generate the VH domain, and human v and j segmentsrecombine to generate the VL domain. The D domain or region refers tothe diversity domain or region of an antibody chain. J domain or regionrefers to the joining domain or region of an antibody chain. Somatichypermutation may result in an antibody VH or VL domain having frameworkregions that do not exactly match or align with the corresponding genesegments, but sequence alignment can be used to identify the closestgene segments and thus identify from which particular combination ofgene segments a particular VH or VL domain is derived. When aligningantibody sequences with gene segments, the antibody amino acid sequencemay be aligned with the amino acid sequence encoded by the gene segment,or the antibody nucleotide sequence may be aligned directly with thenucleotide sequence of the gene segment. Germline gene segmentscorresponding to framework regions of example antibodies describedherein are indicated in Table S-12.

An antibody may be a whole immunoglobulin, including constant regions,or may be an antibody fragment. An antibody fragment is a portion of anintact antibody, for example comprising the antigen binding and/orvariable region of the intact antibody. The antibody fragment mayinclude one or more constant region domains.

An antibody of the invention may be a human antibody or a chimaericantibody comprising human variable regions and non-human (e.g., mouse)constant regions. The antibody of the invention for example has humanvariable regions, and optionally also has human constant regions.

Thus, antibodies optionally include constant regions or parts thereof,e.g., human antibody constant regions or parts thereof, such as a humanIgG4 constant region. For example, a VL domain may be attached at itsC-terminal end to antibody light chain kappa or lambda constant domains.Similarly, an antibody VH domain may be attached at its C-terminal endto all or part (e.g. a CH1 domain or Fc region) of an immunoglobulinheavy chain constant region derived from any antibody isotype, e.g. IgG,IgA, IgE and IgM and any of the isotype sub-classes, such as IgG1 orIgG4.

Digestion of whole (bivalent) immunoglobulins with the enzyme papainresults in two identical (monovalent) antigen-binding fragments known as“Fab” fragments, and an “Fc” fragment. The Fc has no antigen-bindingactivity but has the ability to crystallize. “Fab” when used hereinrefers to a fragment of an antibody that includes one constant and onevariable domain of each of the heavy and light chains. The term “Fcregion” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. The “Fc fragment” refers to the carboxy-terminalportions of both H chains held together by disulphides.

Digestion of antibodies with the enzyme pepsin results in a bivalentF(ab′)2 fragment in which the two arms of the antibody molecule remainlinked. The F(ab′)2 fragment is a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region.Single-chain antibodies (e.g., scFv) are another fragment. Two differentmonovalent monospecific antibody fragments such as scFv may be linkedtogether to form a bivalent bispecific antibody.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites. This regionconsists of a dimer of one heavy and one light chain variable domain intight, non-covalent or covalent association. It is in this configurationthat the three CDRs of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six CDRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree CDRs specific for an antigen) has the ability to recognise andbind antigen, although usually at a lower affinity than the entirebinding site.

Preferably, the bispecific antibody is a dual binding antibody, i.e., abispecific antibody in which both antigen binding domains are formed bya VH/VL pair. Dual binding antibodies include FIT-Ig (see WO2015/103072,incorporated herein by reference), mAb-dAb, dock and lock, Fab-armexchange, SEEDbody, Triomab, LUZ-Y, Fcab, Kλ-body, orthogonal Fab,scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv,intrabody, BITE, diabody, DART, TandAb, scDiabody, scDiabody-CH3,Diabody-CH3, Triple body, Miniantibody, minibody, scFv-CH3 KIH,scFv-CH-CL-scFv, F(ab′)2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab,ImmTAC, knobs-in-holes, knobs-in-holes with common light chain,knobs-in-holes with common light chain and charge pairs, charge pairs,charge pairs with common light chain, DT-IgG, DutaMab, IgG(H)-scFv,scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG,IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv and scFv4-Ig.

In one embodiment, the bispecific antibody is a bispecific IgGcomprising a FIXa-binding polypeptide arm and a FX-binding polypeptidearm, each polypeptide arm comprising a heavy chain and a light chain.The IgG is a tetrameric immunoglobulin comprising

-   -   a first pair of antibody heavy and light chains (heavy-light        chain pair) comprising a FIXa binding Fv region,    -   a second heavy-light chain pair comprising a FX binding Fv        region,    -   wherein each heavy chain comprises a VH domain and a constant        region, and each light chain comprises a VL domain and a        constant region, and wherein the first and second heavy-light        chain pairs associate through heterodimerisation of their heavy        chain constant regions to form the immunoglobulin tetramer.

Optionally, the two polypeptide arms comprise a common light chain, sothe light chain of the first and second heavy-light chain pairs has anidentical amino acid sequence (FIG. 5). Alternatively the twopolypeptide arms may comprise different light chains.

Bispecific antibody may be monovalent for binding FIXa and for bindingFX.

Antibody Constant Regions

As discussed above, antibodies can be provided in various isotypes andwith different constant regions. The Fc region of antibodies isrecognised by Fc receptors and determines the ability of the antibody tomediate cellular effector functions, including antibody-dependentcell-mediated cytotoxicity (ADCC) activity, complement dependentcytotoxicity (CDC) activity and antibody-dependent cell phagocytosis(ADCP) activity. These cellular effector functions involve recruitmentof cells bearing Fc receptors to the site of the target cells, resultingin killing of the antibody-bound cell.

In the context of the present invention it is desirable to avoidcellular effector functions such as ADCC, ADCP and/or CDC. Therefore,bispecific antigen-binding molecules according to the present inventionmay lack Fc effector function, for example they may contain Fc regionsthat do not mediate ADCC, ADCP and/or CDC, or they may lack Fc regionsor lack antibody constant regions entirely. An antibody may have aconstant region which is effector null.

An antibody may have a heavy chain constant region that binds one ormore types of Fc receptor but does not induce cellular effectorfunctions, i.e., does not mediate ADCC, CDC or ADCP activity. Such aconstant region may be unable to bind the particular Fc receptor(s)responsible for triggering ADCC, CDC or ADCP activity.

An antibody may have a heavy chain constant region that does not bindFcγ receptors, for example the constant region may comprise a Leu235Glumutation (i.e., where the wild type leucine residue is mutated to aglutamic acid residue), which may be referred to as an “E” mutation,e.g., IgG4-E. Another optional mutation for a heavy chain constantregion is Ser228Pro (“P” mutation), which increases stability byreducing Fab arm exchange. A heavy chain constant region may be an IgG4comprising both the Leu235Glu mutation and the Ser228Pro mutation (EUnumbering). This “IgG4-PE” heavy chain constant region is effector null.An alternative effector null human constant region is a disabled IgG1.

Antibody constant regions may be engineered to have an extended halflife in vivo. Examples include “YTE” mutations and other half-lifeextending mutations (Dall'Acqua, Kiener & Wu, JBC 281(33):23514-235242006 and WO02/060919, incorporated by reference herein). The triplemutation YTE is a substitution of 3 amino acids in the IgG CH2 domain,these mutations providing tyrosine at residue 252, threonine at residue254 and glutamic acid at residue 256, numbered according to the EU indexof Kabat. As described in the referenced publications, the YTEmodification increases the half-life of the antibody compared with thehalf-life of a corresponding antibody having a human CH2 wild typedomain. To provide an increased duration of efficacy in vivo, antibodiesof the present invention may include antibody constant regions (e.g.,IgG constant regions, e.g., IgG CH2 domains) that have one or moremutations that increase the half life of the antibody compared with thecorresponding wild type human constant region (e.g., IgG, e.g., IgG CH2domain). Half-life may be determined by standard methods, such as aredescribed in WO02/060919.

In some embodiments, a gamma-carboxyglutamic acid-rich (Gla) domain orother membrane-binding domain is included in the bispecific antibody(e.g., at the C terminus of the Fc), to promote localisation of theantibody to the phospholipid membrane at the platelet surface (viainteraction between the Gla domain and the membrane), thereby increasingthe local concentration of bispecific antibody where FIX and FX arenaturally present in vivo. WO2018/145125 described a FVIII mimeticprotein comprising a FIX/FX bispecific antibody and a membrane bindingdomain, e.g., a platelet binding domain such as a C1, C2 domain, a PHdomain, a GLA domain or a membrane binding domain of a platelet membraneglycoprotein. As described therein, the membrane-binding domain may belinked to the C terminal of one or both of the heavy chain constantdomains of the bispecific antibody. Bispecific antigen binding moleculesof the present invention may optionally include the features andmolecular formats described in WO2018/145125.

As discussed below, in bispecific IgG formats or other antibody formatswhere the different antigen binding arms are heterodimerised viaconstant regions, the constant regions may be engineered to promoteheterodimer formation over homodimer formation and/or to facilitatepurification of heterodimers from a mixture of different species.

The anti-FIXaxFX bispecific antibody emicizumab contains a heavy chainconstant region which includes features designed to promote itsassembly, purification and/or therapeutic performance. A bispecificantibody according to the present invention may comprise any one or moreof these features. Thus it may comprise a human IgG4 (e.g., IgHG4*03)heavy chain constant region amino acid sequence comprising one or moreof the following changes (EU numbering):

-   -   Lys196Gln in CH1;    -   Ser228Pro in the hinge region (P mutation);    -   Phe296Tyr in the DE turn of CH2;    -   Glu356Lys in CH3;    -   Lys439Glu in CH3;    -   Leu445Pro in CH3;    -   Deletion of Gly446;    -   Deletion of Lys447.

One each of the mutations Glu356Lys and Lys439Glu are included in thetwo oppositely paired heavy chain constant regions within the Fc of theheterodimeric bispecific, i.e., one heavy chain constant regioncomprises Glu356 and Lys439Glu and the other heavy chain constant regioncomprises Glu356Lys and Lys439 (see the discussion on charge pairingbelow).

A bispecific antibody according to the present invention may comprise anFc region that has any one or more of the features that are present inthe Fc region of emicizumab. It may comprise the Fc region ofemicizumab. In one embodiment, the amino acid sequences of the heavychain constant regions are the amino acid sequences of the emicizumabheavy chain constant regions.

Example amino acid sequences for heavy chain constant regions are shownin Table S-100.

Engineering of Bispecific Antibodies to Facilitate Heterodimer Formationand/or Purification

One of the difficulties with using bispecific antibodies in the clinichas historically been the difficulty of producing them in largequantities and at pharmaceutical grade purity. The “traditional”bispecific IgG format comprises two different pairs of heavy and lightchains, thus 4 different polypeptide chains, which if expressed togethercould assemble into 10 different potential antibody molecules. Themixture of species will include homodimers (homodimeric anti-FIXabinding arms and homodimeric anti-FX binding arms), molecules in whichone or both light chains are swapped between the H-L pairs, as well asthe “correct” bispecific heterodimeric structure.

Alternative molecular formats have been developed which avoid thispotential mis-pairing, and several examples are provided herein. Theseinclude F(ab′)2, e.g., prepared by chemical coupling or leucine zipper(fos:jun) assembly, diabodies, and scFv heterodimers. Nevertheless, itremains desirable to be able to use bispecific IgG, to reflect thenative structure of antibodies in the bloodstream and to minimiseimmunogenicity of the administered therapeutic molecule. Additionally, afull length bispecific antibody may have a longer serum half-life.

“Knobs into holes” technology for making bispecific antibodies wasdescribed in [14] and in U.S. Pat. No. 5,731,168, both incorporatedherein by reference. The principle is to engineer paired CH3 domains ofheterodimeric heavy chains so that one CH3 domain contains a “knob” andthe other CH3 domains contains a “hole” at a sterically oppositeposition. Knobs are created by replacing small amino acid side chain atthe interface between the CH3 domains, while holes are created byreplacing large side chains with smaller ones. The knob is designed toinsert into the hole, to favour heterodimerisation of the different CH3domains while destabilising homodimer formation. In in a mixture ofantibody heavy and light chains that assemble to form a bispecificantibody, the proportion of IgG molecules having paired heterodimericheavy chains is thus increased, raising yield and recovery of the activemolecule

Mutations Y349C and/or T366W may be included to form “knobs” in an IgGCH3 domain. Mutations E356C, T366S, L368A and/or Y407V may be includedto form “holes” in an IgG CH3 domain. Knobs and holes may be introducedinto any human IgG CH3 domain, e.g., an IgG1, IgG2, IgG3 or IgG4 CH3domain. A preferred example is IgG4. As noted, the IgG4 may includefurther modifications such as the “P” and/or “E” mutations. An exampleIgG4-PE sequence and other example constant regions includingknobs-into-holes mutations are shown in Table S-100. The IgG4 type a(“ra”) sequence contains substitutions Y349C and T366W (“knobs”), andthe IgG4 type b (“yb”) sequence contains substitutions E356C, T366S,L368A, and Y407V (“holes”). Both ra and yb also contain the “P”substitution at position 228 in the hinge (S228P), to stabilise thehinge region of the heavy chain. Both ra and yb also contain the “E”substitution in the CH2 region at position 235 (L235S), to abolishbinding to FcγR. Thus the relevant sequence of the IgG4-PE heavy chainis ppcpPcpapefEggps (SEQ ID NO: 401). A bispecific antigen bindingmolecule of the present invention may contain an IgG4 PE human heavychain constant region (e.g., SEQ ID NO: 143), optionally two such pairedconstant regions, optionally wherein one has “knobs” mutations and onehas “holes” mutations, e.g., wherein one heavy chain constant region hasa sequence SEQ ID NO: 144 (knobs) and one heavy chain constant regionhas a sequence SEQ ID NO: 145 (holes).

A further advance in bispecific IgG engineering was the idea of using acommon light chain, as described in WO98/50431. Bispecific antibodiescomprising two heavy-light chain pairs were described, in which thevariable light chains of both heavy-light chain pairs had a commonsequence. WO98/50431 described combining the common light chain approachwith specific complementary interactions in the heavy chainheterodimerisation interface (such as knobs-into-holes) to promoteheterodimer formation and hinder homodimer formation. In combination,these approaches enhance formation of the desired heterodimer relativeto undesired heterodimers and homodimers.

While knobs-into-holes technology involves engineering amino acid sidechains to create complementary molecular shapes at the interface of thepaired CH3 domains in the bispecific heterodimer, another way to promoteheterodimer formation and hinder homodimer formation is to engineer theamino acid side chains to have opposite charges. Association of CH3domains in the heavy chain heterodimers is favoured by the pairing ofoppositely charged residues, while paired positive charges or pairednegative charges would make homodimer formation less energeticallyfavourable. WO2006/106905 described a method for producing aheteromultimer composed of more than one type of polypeptide (such as aheterodimer of two different antibody heavy chains) comprising asubstitution in an amino acid residue forming an interface between saidpolypeptides such that heteromultimer association will be regulated, themethod comprising:

-   -   (a) modifying a nucleic acid encoding an amino acid residue        forming the interface between polypeptides from the original        nucleic acid, such that the association between polypeptides        forming one or more multimers will be inhibited in a        heteromultimer that may form two or more types of multimers;    -   (b) culturing host cells such that a nucleic acid sequence        modified by step (a) is expressed; and    -   (c) recovering said heteromultimer from the host cell culture,    -   wherein the modification of step (a) is modifying the original        nucleic acid so that one or more amino acid residues are        substituted at the interface such that two or more amino acid        residues, including the mutated residue(s), forming the        interface will carry the same type of positive or negative        charge.

An example of this is to suppress association between heavy chains byintroducing electrostatic repulsion at the interface of the heavy chainhomodimers, for example by modifying amino acid residues that contacteach other at the interface of the CH3 domains, including:

-   -   positions 356 and 439    -   positions 357 and 370    -   positions 399 and 409,    -   the residue numbering being according to the EU numbering        system.

By modifying one or more of these pairs of residues to have like charges(both positive or both negative) in the CH3 domain of a first heavychain, the pairing of heavy chain homodimers is inhibited byelectrostatic repulsion. By engineering the same pairs or pairs ofresidues in the CH3 domain of a second (different) heavy chain to havean opposite charge compared with the corresponding residues in the firstheavy chain, the heterodimeric pairing of the first and second heavychains is promoted by electrostatic attraction.

Amino acids at the heavy chain constant region CH3 interface weremodified to introduce charge pairs, the mutations being listed in Table1 of WO2006/106905. It was reported that modifying the amino acids atheavy chain positions 356, 357, 370, 399, 409 and 439 to introducecharge-induced molecular repulsion at the CH3 interface had the effectof increasing efficiency of formation of the intended bispecificantibody. For example, one heavy chain constant region may be an IgG4constant region containing mutation K439E (positively charged Lysreplaced by negatively charged Glu) and the other heavy chain constantregion may be an IgG4 constant region containing mutation E356K(negatively charged Glu replaced by positively charged Lys), using EUnumbering. “Charge pairing” results from spatial proximity of residues439 and 356 in an Fc region assembled from heterodimerisation of thesetwo constant regions.

Where two different heavy chain constant regions are used, these may beconnected to the two different VH domains of the antibody in eitherorientation. For example,

-   -   a first heavy chain may comprise an anti-FIX VH domain and a        constant region comprising K439E, and a second heavy chain may        comprise an anti-FX VH domain and a constant region comprising        E356K, or    -   a first heavy chain may comprise an anti-FIX VH domain and a        constant region comprising E356K, and a second heavy chain may        comprise an anti-FX VH domain and a constant region comprising        K439E.

WO2006/106905 also exemplified bispecific IgG antibodies binding FX andFIXa in which the CH3 domains of IgG4 were engineered withknobs-into-holes mutations. Type a Type a (IgG4γa) was an IgG4substituted at Y349C and T366W, and type b (IgG4γb) was an IgG4substituted at E356C, T366S, L368A, and Y407V.

In another example, introduction of charge pairs in the antibody VH andVL domains was used to inhibit the formation of “incorrect” VH-VL pairs(pairing of VH from one antibody with VL of the other antibody). In oneexample, Q residues in the VH and VL were changed to K or R (positive),or to E or D (negative), to inhibit hydrogen bonding between the Q sidechains and to introduce electrostatic repulsion.

Further examples of charge pairs were disclosed in WO2013/157954, whichdescribed a method for producing a heterodimeric CH3 domain-comprisingmolecule from a single cell, the molecule comprising two CH3 domainscapable of forming an interface. The method comprised providing in thecell

-   -   (a) a first nucleic acid molecule encoding a first CH3        domain-comprising polypeptide chain, this chain comprising a K        residue at position 366 according to the EU numbering system and    -   (b) a second nucleic acid molecule encoding a second CH3        domain-comprising polypeptide chain, this chain comprising a D        residue at position 351 according to the EU numbering system,    -   the method further comprising the step of culturing the host        cell, allowing expression of the two nucleic acid molecules and        harvesting the heterodimeric CH3 domain-comprising molecule from        the culture.

Further methods of engineering electrostatic interactions in polypeptidechains to promote heterodimer formation over homodimer formation weredescribed in WO2011/143545.

Another example of engineering at the CH3-CH3 interface isstrand-exchange engineered domain (SEED) CH3 heterodimers. The CH3domains are composed of alternating segments of human IgA and IgG CH3sequences, which form pairs of complementary SEED heterodimers referredto as “SEED-bodies” [15; WO2007/110205].

Bispecifics have also been produced with heterodimerised heavy chainsthat are differentially modified in the CH3 domain to alter theiraffinity for binding to a purification reagent such as Protein A.WO2010/151792 described a heterodimeric bispecific antigen-bindingprotein comprising

-   -   a first polypeptide comprising, from N-terminal to C-terminal, a        first epitope-binding region that selectively binds a first        epitope, an immunoglobulin constant region that comprises a        first CH3 region of a human IgG selected from IgG1, IgG2, and        IgG4; and    -   a second polypeptide comprising, from N-terminal to C-terminal,        a second epitope-binding region that selectively binds a second        epitope, an immunoglobulin constant region that comprises a        second CH3 region of a human IgG selected from IgG1, IgG2, and        IgG4, wherein the second CH3 region comprises a modification        that reduces or eliminates binding of the second CH3 domain to        Protein A.

The Fc region may thus comprise one or more mutations to promotedifferential purification of the active heterodimer from homodimerspecies. The CH3 of one heavy chain constant region may comprise themutation His435Arg and/or Tyr436Phe (EU numbering) [16] while the CH3 ofthe other heavy chain constant region lacks said mutations. Emicizumab,for example, comprises an Fc region in which one CH3 comprises His435and the other CH3 comprises His435Arg.

The bispecifics of the present invention may employ any of thesetechniques and molecular formats as desired.

Generating and Modifying Antibodies

Methods for identifying and preparing antibodies are well known.Isolated (optionally mutated) nucleic acid encoding antibodies (orheavy-light chain pairs or polypeptide binding arms thereof) describedherein may be introduced into host cells, e.g., CHO cells as discussed.Host cells are then cultured under conditions for expression of theantibody (or of the antibody heavy and/or light chain variable domain,heavy-light chain pair, or polypeptide binding arm) to produce thedesired antibody format. Some possible antibody formats are describedherein, e.g., whole immunoglobulins, antigen-binding fragments, andother designs.

Variable domain amino acid sequence variants of any of the VH and VLdomains or CDRs whose sequences are specifically disclosed herein andmay be employed in accordance with the present invention, as discussed.

Alterations to nucleic acid encoding the antibody heavy and/or lightchain variable domain may be performed, such as mutation of residues andgeneration of variants, as described herein. There are many reasons whyit may be desirable to create variants, which include optimising theantibody sequence for large-scale manufacturing, facilitatingpurification, enhancing stability or improving suitability for inclusionin a desired pharmaceutical formulation. Protein engineering work can beperformed at one or more target residues in the antibody sequence, e.g.,to substituting one amino acid with an alternative amino acid(optionally, generating variants containing all naturally occurringamino acids at this position, with the possible exception of Cys andMet), and monitoring the impact on function and expression to determinethe best substitution. It is in some instances undesirable to substitutea residue with Cys or Met, or to introduce these residues into asequence, as to do so may generate difficulties in manufacturing—forinstance through the formation of new intramolecular or intermolecularcysteine-cysteine bonds. Where a lead candidate has been selected and isbeing optimised for manufacturing and clinical development, it willgenerally be desirable to change its antigen-binding properties aslittle as possible, or at least to retain the affinity and potency ofthe parent molecule. However, variants may also be generated in order tomodulate key antibody characteristics such as affinity, cross-reactivityor neutralising potency.

One or more amino acid mutations may optionally be made in frameworkregions of an antibody VH or VL domain disclosed herein. For example,one or more residues that differ from the corresponding human germlinesegment sequence may be reverted to germline. Human germline genesegment sequences corresponding to VH and VL domains of exampleantibodies herein are indicated in Table S-12.

In a bispecific antigen binding molecule, an antigen-binding site maycomprise a set of H and/or L CDRs of any of the disclosed anti-FIX oranti-FX antibodies with one or more amino acid mutations within thedisclosed set of H and/or L CDRs. The mutation may be an amino acidsubstitution, deletion or insertion. Thus for example there may be oneor more amino acid substitutions within the disclosed set of H and/or LCDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or2 mutations e.g. substitutions, within the set of H and/or L CDRs. Forexample, there may be up to 6, 5, 4, 3 or 2 mutations, e.g.substitutions, in HCDR3 and/or there may be up to 6, 5, 4, 3, or 2mutations, e.g. substitutions, in LCDR3.

An antibody may comprise a VH domain that has at least 60, 70, 80, 85,90, 95, 98 or 99 amino acid sequence identity with a VH domain as shownin the Tables, and/or comprising a VL domain that has at least 60, 70,80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domainof any of those antibodies. Algorithms that can be used to calculate %identity of two amino acid sequences include e.g. BLAST, FASTA, or theSmith-Waterman algorithm, e.g. employing default parameters. Particularvariants may include one or more amino acid sequence alterations(addition, deletion, substitution and/or insertion of an amino acidresidue).

Alterations may be made in one or more framework regions and/or one ormore CDRs. Variants are optionally provided by CDR mutagenesis. Thealterations normally do not result in loss of function, so an antibodycomprising a thus-altered amino acid sequence may retain an ability tobind its antigen. It may retain the same quantitative binding ability asan antibody in which the alteration is not made, e.g. as measured in anassay described herein. The antibody comprising a thus-altered aminoacid sequence may have an improved ability to bind its antigen.

Alteration may comprise replacing one or more amino acid residue with anon-naturally occurring or non-standard amino acid, modifying one ormore amino acid residue into a non-naturally occurring or non-standardform, or inserting one or more non-naturally occurring or non-standardamino acid into the sequence. Examples of numbers and locations ofalterations in sequences of the invention are described elsewhereherein. Naturally occurring amino acids include the 20 “standard”L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C,K, R, H, D, E by their standard single-letter codes. Non-standard aminoacids include any other residue that may be incorporated into apolypeptide backbone or result from modification of an existing aminoacid residue. Non-standard amino acids may be naturally occurring ornon-naturally occurring.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from a parent polypeptide or nucleic acid by one or moreamino acid or nucleic acid deletions, substitutions or additions, yetretains one or more specific functions or biological activities of theparent molecule. Amino acid substitutions include alterations in whichan amino acid is replaced with a different naturally-occurring aminoacid residue. Such substitutions may be classified as “conservative”, inwhich case an amino acid residue contained in a polypeptide is replacedwith another naturally occurring amino acid of similar character eitherin relation to polarity, side chain functionality or size. Suchconservative substitutions are well known in the art. Substitutionsencompassed by the present invention may also be “non-conservative”, inwhich an amino acid residue which is present in a peptide is substitutedwith an amino acid having different properties, such asnaturally-occurring amino acid from a different group (e.g.,substituting a charged or hydrophobic amino; acid with alanine), oralternatively, in which a naturally-occurring amino acid is substitutedwith a non-conventional amino acid. In some embodiments amino acidsubstitutions are conservative. Also encompassed within the term variantwhen used with reference to a polynucleotide or polypeptide, refers to apolynucleotide or polypeptide that can vary in primary, secondary, ortertiary structure, as compared to a reference polynucleotide orpolypeptide, respectively (e.g., as compared to a wild-typepolynucleotide or polypeptide).

In some aspects, one can use “synthetic variants”, “recombinantvariants”, or “chemically modified” polynucleotide variants orpolypeptide variants isolated or generated using methods well known inthe art. “Modified variants” can include conservative ornon-conservative amino acid changes, as described below. Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Some aspects include insertion variants, deletion variants orsubstituted variants with substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. The term “conservativesubstitution,” when describing a polypeptide, refers to a change in theamino acid composition of the polypeptide that does not substantiallyalter the polypeptide's activity. For example, a conservativesubstitution refers to substituting an amino acid residue for adifferent amino acid residue that has similar chemical properties (e.g.,acidic, basic, positively or negatively charged, polar or nonpolar,etc.). Conservative amino acid substitutions include replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, ora threonine with a serine. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. For example,the following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (VV). (See also Creighton, Proteins, W. H. Freeman andCompany (1984), incorporated by reference in its entirety.) In someembodiments, individual substitutions, deletions or additions thatalter, add or delete a single amino acid or a small percentage of aminoacids can also be considered “conservative substitutions” if the changedoes not reduce the activity of the peptide. Insertions or deletions aretypically in the range of about 1 to 5 amino acids. The choice ofconservative amino acids may be selected based on the location of theamino acid to be substituted in the peptide, for example if the aminoacid is on the exterior of the peptide and expose to solvents, or on theinterior and not exposed to solvents.

One can select the amino acid that will substitute an existing aminoacid based on the location of the existing amino acid, including itsexposure to solvents (i.e., if the amino acid is exposed to solvents oris present on the outer surface of the peptide or polypeptide ascompared to internally localized amino acids not exposed to solvents).Selection of such conservative amino acid substitutions are well knownin the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999,217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S.French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one canselect conservative amino acid substitutions suitable for amino acids onthe exterior of a protein or peptide (i.e. amino acids exposed to asolvent), for example, but not limited to, the following substitutionscan be used: substitution of Y with F, T with S or K, P with A, E with Dor Q, N with D or G, R with K, G with N or A, T with S or K, D with N orE, I with L or V, F with Y, S with T or A, R with K, G with N or A, Kwith R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

The invention includes methods of producing polypeptide binding armscontaining VH and/or VL domain variants of the antibody VH and/or VLdomains shown in the Tables herein. FIXa binding polypeptide armscomprising variant VH domains may be produced by a method comprising

-   -   (i) providing, by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of a parent antibody VH domain, an antibody VH domain that is an        amino acid sequence variant of the parent antibody VH domain,    -   wherein the parent antibody VH domain is a VH domain shown in        FIG. 20, e.g., N1280H, or is a VH domain comprising the heavy        chain complementarity determining regions of any of those VH        domains,    -   (ii) optionally combining the VH domain thus provided with a VL        domain, to provide a VH/VL combination, and    -   (iii) testing the VH domain or VH/VL domain combination thus        provided to identify an antibody with one or more desired        characteristics.

The VH domain may be any VH domain whose sequence is shown in Table S-9Aor FIG. 20, or any VH domain comprising a set of HCDRs (HCDR1, HCDR2 andHCDR3) of a VH domain shown in Table S-9A or FIG. 20.

Desired characteristics of FIXa-binding polypeptide arms, and ofbispecific anti-FIXa/FX binding molecules comprising them, are detailedelsewhere herein. For example, the method may comprise confirming thatthe VH domain or VH/VL domain combination binds FIXa as describedherein.

When VL domains are included in the method, the VL domain may be theN0128L VL domain or may be a variant provided by way of addition,deletion, substitution or insertion of one or more amino acids in theamino acid sequence of the N0128L VL domain, or may be a VL domaincomprising the light chain complementarity determining regions of theN0128L VL domain. The VL domain may be the 0325L VL domain.

Methods of generating variant antibodies may optionally compriseproducing copies of the antibody or VH/VL domain combination. Methodsmay further comprise producing a bispecific antibody comprising the FIXabinding polypeptide arm, for example by expression of encoding nucleicacid. Suitable methods of expression, including recombinant expressionin host cells, are set out in detail herein.

Encoding Nucleic Acids and Methods of Expression

Isolated nucleic acid may be provided, encoding bispecific antigenbinding molecules, e.g., bispecific antibodies, according to the presentinvention. Nucleic acid may be DNA and/or RNA. Genomic DNA, cDNA, mRNAor other RNA, of synthetic origin, or any combination thereof can encodean antibody.

The present invention provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above. Exemplary nucleotide sequences are includedin the Tables. Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesa RNA molecule with the specified sequence in which U is substituted forT, unless context requires otherwise.

The present invention also provides a recombinant host cell thatcomprises one or more nucleic acids encoding the antigen bindingmolecule. Methods of producing the encoded molecule may compriseexpression from the nucleic acid, e.g., by culturing recombinant hostcells containing the nucleic acid. The bispecific molecule may thus beobtained, and may be isolated and/or purified using any suitabletechnique, then used as appropriate. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cellsis well established in the art. A common bacterial host is E. coli.Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production. Mammalian cell linesavailable in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, humanembryonic kidney cells, human embryonic retina cells and many others.

Vectors may contain appropriate regulatory sequences, including promotersequences, terminator sequences, polyadenylation sequences, enhancersequences, marker genes and other sequences as appropriate. Nucleic acidencoding an antibody can be introduced into a host cell. Nucleic acidcan be introduced to eukaryotic cells by various methods, includingcalcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using retrovirus orother virus, e.g. vaccinia or, for insect cells, baculovirus.Introducing nucleic acid in the host cell, in particular a eukaryoticcell may use a viral or a plasmid based system. The plasmid system maybe maintained episomally or may be incorporated into the host cell orinto an artificial chromosome. Incorporation may be either by random ortargeted integration of one or more copies at single or multiple loci.For bacterial cells, suitable techniques include calcium chloridetransformation, electroporation and transfection using bacteriophage.The introduction may be followed by expressing the nucleic acid, e.g.,by culturing host cells under conditions for expression of the gene,then optionally isolating or purifying the antibody.

Nucleic acid of the invention may be integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences that promote recombination with the genome, in accordancewith standard techniques. Nucleic acid encoding a bispecific may beintegrated into genomic DNA of a host (e.g., CHO) cell, e.g., intochromosomal DNA, and the resulting recombinant cell may be cultured toexpress the bispecific. A cell line development process may compriseintroducing nucleic acid encoding the bispecific into multiple hostcells, and selecting a cell line which expresses a desired level ofbispecific antibody (e.g., at least 95% heterodimer, with no more than5% homodimeric contaminants) at the desired yield (e.g., at least 0.5g/L or at least 1 g/L). Preferably the cell line will retain stableexpression over a number of generations in cell culture, and thus it maymaintain these levels of production over a at least 60 generations forexample.

The present invention also provides a method that comprises usingnucleic acid described herein in an expression system in order toexpress the bispecific antigen binding molecule. Desirably, theantigen-binding molecules are expressed at a yield of at least 0.5 g/Lin the cell supernatant after initial fermentation, preferably at ayield of >2 g/L. Solubility should be >10 mg/ml, preferably >50 mg/ml,without significant aggregation or degradation of the molecules.

To provide medicines suitable for global treatment, antibodies can beproduced on a large scale, for instance in cell culture volumes of atleast 100 litres or at least 200 litres, e.g., between 100-250 litres.Batch culture, particularly fed-batch culture, is now commonly used forproduction of biotherapeutics for clinical and commercial use, and suchmethods may suitably be used in the present invention to generate theantibodies, followed by purification and formulation steps as notedherein. Bioreactors may be metal (e.g., stainless steel) vessels or maybe single-use bioreactors.

Formulation and Administration

The bispecific antigen-binding molecules (“bispecifics”) according tothe present invention, and their encoding nucleic acid molecules, willusually be provided in isolated form. The bispecifics VH and/or VLdomains, and nucleic acids may be provided purified from their naturalenvironment or their production environment. Isolated antigen-bindingmolecules and isolated nucleic acid will be free or substantially freeof material with which they are naturally associated, such as otherpolypeptides or nucleic acids with which they are found in vivo, or theenvironment in which they are prepared (e.g., cell culture) when suchpreparation is by recombinant DNA technology in vitro. Optionally anisolated antigen-binding molecule or nucleic acid (1) is free of atleast some other proteins with which it would normally be found, (2) isessentially free of other proteins from the same source, e.g., from thesame species, (3) is expressed by a cell from a different species, (4)has been separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is associated innature, (5) is operably associated (by covalent or noncovalentinteraction) with a polypeptide with which it is not associated innature, or (6) does not occur in nature.

Bispecific antibody may be purified (e.g., from cell culturesupernatant) by protein A chromatography and/or ion exchangechromatography. The bispecific antibody may be produced by a methodcomprising

-   -   expressing two antibody heavy chains and common light chain from        cultured host cells comprising encoding nucleic acids,    -   obtaining cell culture comprising the bispecific antibody and        monospecific antibodies assembled from the antibody heavy chains        and common light chain,    -   isolating the bispecific antibody and monospecific antibodies        from the cell culture (e.g., using protein A chromatography),        and    -   purifying the bispecific antibody from the monospecific        antibodies (e.g., using cation exchange chromatography).

Bispecifics or their encoding nucleic acids may be formulated withdiluents or adjuvants and still for practical purposes be isolated—forexample they may be mixed with carriers if used to coat microtitreplates for use in immunoassays, and may be mixed with pharmaceuticallyacceptable carriers or diluents when used in therapy. As describedelsewhere herein, other active ingredients may also be included intherapeutic preparations. The antigen binding molecules may beglycosylated, either naturally in vivo or by systems of heterologouseukaryotic cells such as CHO cells, or they may be (for example ifproduced by expression in a prokaryotic cell) unglycosylated. Theinvention encompasses antibodies having a modified glycosylationpattern.

Typically, an isolated product constitutes at least about 5%, at leastabout 10%, at least about 25%, or at least about 50% of a given sample.A bispecific may be substantially free from proteins or polypeptides orother contaminants that are found in its natural or productionenvironment that would interfere with its therapeutic, diagnostic,prophylactic, research or other use.

As discussed elsewhere herein, expression of antibody heavy and lightchains for a bispecific antibody may generate unwanted homodimericspecies (anti-FIX and anti-FX antibodies) in addition to the activeheterodimeric bispecific antibody. Preferably a bispecific is providedin a composition in which the heterodimeric bispecific antibody isrepresents at least 95% of the total antibody, with homodimeric antibodycontaminants being present at 5% or less. The composition may compriseat least 98% or at least 99% heterodimeric bispecific, with homodimericcontaminants representing 0-2% or 0-1% respectively.

The invention provides therapeutic compositions comprising thebispecifics described herein. Therapeutic compositions comprisingnucleic acid encoding such bispecifics are also provided. Encodingnucleic acids are described in more detail elsewhere herein and includeDNA and RNA, e.g., mRNA. In therapeutic methods described herein, use ofnucleic acid encoding the bispecific, and/or of cells containing suchnucleic acid, may be used as alternatives (or in addition) tocompositions comprising the bispecific molecule itself. Cells containingnucleic acid encoding the bispecific, optionally wherein the nucleicacid is stably integrated into the genome, thus represent medicamentsfor therapeutic use in a patient. Nucleic acid encoding the bispecificmay be introduced into human cells derived from the intended patient andmodified ex vivo. Administration of cells containing the encodingnucleic acid to the patient provides a reservoir of cells capable ofexpressing the bispecific, which may provide therapeutic benefit over alonger term compared with administration of isolated nucleic acid or theisolated bispecific molecule. Nucleic acid encoding the bispecific maybe provided for use in gene therapy, comprising introducing the encodingnucleic acid into cells of the patient in vivo, so that the nucleic acidis expressed in the patient's cells and provides a therapeutic effectsuch as compensating for hereditary factor VIII deficiency.

Compositions may contain suitable carriers, excipients, and other agentsthat are incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311. Compositions may comprise the antibody ornucleic acid in combination with medical injection buffer.

Bispecifics, or their encoding nucleic acids, may be formulated for thedesired route of administration to a patient, e.g., in liquid(optionally aqueous solution) for injection. An example buffer in whichto formulate the bispecific for injection is an aqueous solution of 20mM sodium acetate, 150 mM arginine hydrochloride, 0.05% w/v polysorbate80 pH 5.2.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention. Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Theantigen-binding molecules are preferably administered by subcutaneousinjection.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared can be filled in an appropriate ampoule. A pharmaceuticalcomposition of the present invention can be delivered subcutaneously orintravenously with a standard needle and syringe. It is envisaged thattreatment will not be restricted to use in the clinic. Therefore,subcutaneous injection using a needle-free device is also advantageous.With respect to subcutaneous delivery, a pen delivery device readily hasapplications in delivering a pharmaceutical composition of the presentinvention. Such a pen delivery device can be reusable or disposable. Areusable pen delivery device generally utilizes a replaceable cartridgethat contains a pharmaceutical composition. Once all of thepharmaceutical composition within the cartridge has been administeredand the cartridge is empty, the empty cartridge can readily be discardedand replaced with a new cartridge that contains the pharmaceuticalcomposition. The pen delivery device can then be reused. In a disposablepen delivery device, there is no replaceable cartridge. Rather, thedisposable pen delivery device comes prefilled with the pharmaceuticalcomposition held in a reservoir within the device. Once the reservoir isemptied of the pharmaceutical composition, the entire device isdiscarded. Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPENTMI, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, the aforesaid antibody may becontained in about 5 to about 100 mg and in about 10 to about 250 mg forthe other dosage forms.

The bispecific, nucleic acid, or composition comprising it, may becontained in a medical container such as a phial, syringe, IV containeror an injection device. In an example, the bispecific, nucleic acid orcomposition is in vitro, and may be in a sterile container. In anexample, a kit is provided comprising the bispecific, packaging andinstructions for use in a therapeutic method as described herein.

One aspect of the invention is a composition comprising a bispecific ornucleic acid of the invention and one or more pharmaceuticallyacceptable excipients, examples of which are listed above.“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A pharmaceutically acceptable carrier,excipient, or adjuvant can be administered to a patient, together with abispecific agent, e.g., any antibody or polypeptide molecule describedherein, and does not destroy the pharmacological activity thereof and isnontoxic when administered in doses sufficient to deliver a therapeuticamount of the agent.

In some embodiments, the bispecific will be the sole active ingredientin a composition according to the present invention. Thus, a compositionmay consist of the antibody or it may consist of the bispecific with oneor more pharmaceutically acceptable excipients. However, compositionsaccording to the present invention optionally include one or moreadditional active ingredients.

Where required (for example, for management of acute bleeds), thebispecific may be combined with one or more other treatments forhaemophilia, including recombinant factor VIII (e.g., turoctocog alfa)or recombinant factor VIIa (e.g., eptacog alfa). The functionalproperties and safety profile of bispecifics described herein arebelieved to be suitable for their safe combination with such furthertherapeutic agents. The bispecific may be combined with recombinantfactor Va (FVa), for example an activated variant FVa as described inU.S. Ser. No. 10/407,488.

Other therapeutic agents that it may be desirable to administer withbispecific or nucleic acids according to the present invention includeanalgaesic agents. Any such agent or combination of agents may beadministered in combination with, or provided in compositions withantibodies or nucleic acids according to the present invention, whetheras a combined or separate preparation. The bispecific or nucleic acidaccording to the present invention may be administered separately andsequentially, or concurrently and optionally as a combined preparation,with another therapeutic agent or agents such as those mentioned.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

Bispecifics, and their encoding nucleic acids, can be used astherapeutic agents. Patients herein are generally mammals, typicallyhumans. A bispecific or nucleic acid may be administered to a mammal,e.g., by any route of administration mentioned herein.

Administration is normally in a “therapeutically effective amount”, thisbeing an amount that produces the desired effect for which it isadministered, sufficient to show benefit to a patient. The exact amountwill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding). Prescription of treatment, e.g. decisions on dosage etc,is within the responsibility of general practitioners and other medicaldoctors and may depend on the severity of the symptoms and/orprogression of a disease being treated. A therapeutically effectiveamount or suitable dose of bispecific or nucleic acid can be determinedby comparing its in vitro activity and in vivo activity in an animalmodel. Methods for extrapolation of effective dosages in mice and othertest animals to humans are known.

Bispecifics may be administered in an amount in one of the followingranges per dose:

-   -   about 10 μg/kg body weight to about 100 mg/kg body weight,    -   about 50 μg/kg body weight to about 5 mg/kg body weight,    -   about 100 μg/kg body weight to about 10 mg/kg body weight,    -   about 100 μg/kg body weight to about 20 mg/kg body weight,    -   about 0.5 mg/kg body weight to about 20 mg/kg body weight, or    -   about 5 mg/kg body weight or lower, for example less than 4,        less than 3, less than 2, or less than 1 mg/kg of the antibody.

The dose of antigen-binding molecule administered may be up to 1 mg/kg.It may be formulated at lower strength for paediatric populations, forexample 30-150 mg/mL. The bispecific molecule may be packaged in smallerquantities for a paediatric population, e.g., it may be provided inphials of 25-75 mg, e.g., 30 or 60 mg.

In methods of treatment described herein, one or more doses may beadministered. In some cases, a single dose may be effective to achieve along-term benefit. Thus, the method may comprise administering a singledose of the bispecific, its encoding nucleic acid, or the composition.Alternatively, multiple doses may be administered, usually sequentiallyand separated by a period of days, weeks or months. Optionally, thebispecific may be administered to a patient once a month, or lessfrequently, e.g., every two months or every three months.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilised (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well. In the context of the invention, treatmentmay be preventative treatment.

Long half-life is a desirable feature in the bispecifics of the presentinvention. Extended half-life translates to less frequentadministration, with fewer injections being required to maintain atherapeutically effective concentration of the molecule in thebloodstream. The in vivo half life of antigen-binding molecules of thepresent invention in humans may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or 21 days, or longer. The in vivo half life ofantigen-binding molecules in non-human primates such as cynomolgusmonkeys may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21days, or longer.

Maintenance of 1% of normal FVIII activity is considered to be a minimumfor prophylactic use in haemophilia [1]. In a paper reporting a humanclinical trial with ACE910 (emicizumab), in silico populationpharmacokinetic modelling and simulations suggested that a weekly doseof 1 mg/kg resulted in a plasma concentration of at least about 300 nM(45 μg/ml), producing a continuous haemostatic effect of at least 10% ofnormal FVIII activity [4]. This dose was also reported to be welltolerated in patients.

Based on that data, a plasma concentration range of approximately 30 nM(˜4.5 μg/ml) to 300 nM (˜45 μg/ml) would correspond to effective FVIIIactivity of 1-10%, assuming a linear relationship between antibodyconcentration and FVIII activity and comparable antibody activity.

Bispecifics according to the present invention exhibit exceptionallyhigh activity at concentrations in the range of 30 nM (˜4.5 μg/ml) to300 nM (˜45 μg/ml), maintaining strong thrombin generation activity evenat relatively low doses. As evidenced by its high potency (see, e.g.,Example 14), a bispecific antibody according to the present inventionmay exhibit therapeutic efficacy at a lower plasma concentration thanemicizumab. Therefore it may provide greater therapeutic benefit at anequivalent dose, and it may provide equivalent therapeutic benefit at alower dose, compared with emicizumab. Patients can thus benefit fromsmaller and/or less frequent injections, and healthcare providers canbenefit from lower associated costs.

The US FDA currently (in guidance issued October 2018) recommends thatemicizumab be administered at a loading dose of 3 mg/kg by subcutaneousinjection once weekly for the first 4 weeks, followed by a maintenancedose of:

-   -   1.5 mg/kg once every week, or    -   3 mg/kg once every 2 weeks, or    -   6 mg/kg once every 4 weeks.

Antigen-binding molecules according to the present invention may beprovided for administration at regular intervals of one week, two weeks,three weeks, four weeks, or one month.

In a preferred embodiment, the bispecific is administered bysubcutaneous injection.

Therapeutic Use

The bispecific antigen-binding molecules of the present invention may beused in a method of treatment of the human or animal body by therapy.Therapeutic indications for the molecules include:

-   -   use to treat haemophilia A,    -   use to treat hereditary factor VIII deficiency,    -   use to significantly decrease the number of bleeding incidents        in haemophilia A patients,    -   use to substitute for factor VIII function,    -   and/or    -   use to promote blood coagulation.

Patients are typically human patients. The patient may be a humandiagnosed with haemophilia A or hereditary factor VIII deficiency, or ahuman who has lower (or absent) factor VIII expression or activitycompared with wild type. The patient may be a paediatric patient (e.g.,from 2 to less than 18 years of age) or may be an adult. The patient maybe a human male. The patient may or may not have inhibitors to factorVIII.

A bispecific molecule of the present invention, or a compositioncomprising such a bispecific molecule or its encoding nucleic acid, maybe used or provided for use in any such method. Use of the bispecificmolecule, or of a composition comprising it or its encoding nucleicacid, for the manufacture of a medicament for use in any such method isalso envisaged. The method typically comprises administering theantibody or composition to a mammal, e.g., a human patient. Suitableformulations and methods of administration are described elsewhereherein.

There is a presently unmet need for treatment of haemophilia A patientswho develop inhibitory allo-antibodies to FVIII. Antigen-bindingmolecules of the present invention are suitable for use in suchpatients. Accordingly, in some aspects, a patient treated with abispecific antigen binding molecule according to the present inventionmay be resistant to treatment with FVIII owing to the presence ofinhibitory antibodies in the bloodstream. Resistance to treatment can bemanifested in a reduction of efficacy of the therapy. Such resistancemay be detected in in vitro assays (e.g. aPTT assay) with a blood plasmasample from the patient, wherein the therapeutic molecule does notreduce coagulation time to the same level as in an assay with controlFVIII-deficient plasma (the latter lacking inhibitory antibodies to thetherapeutic molecule).

Patients receiving other treatments for haemophilia, such as bispecificantibodies to FIXa and FX, may also develop inhibitory antibodies tothose therapeutic antibodies. As noted, use of human antibodies such asthose of the present invention should minimise the risk of this, butinhibitory antibodies may nevertheless be generated in some patients whoreceive antigen binding molecules of the present invention or otherbispecific antigen binding molecules to FIXa and FX. A patient treatedwith a bispecific antigen binding molecule according to the presentinvention may be resistant to treatment to a different bispecificantigen binding molecule for FIXa and FX owing to the presence ofinhibitory antibodies in the bloodstream. The patient may be resistantto treatment with emicizumab.

Since inhibitory antibodies may be generated through long termtherapeutic administration of a drug product, it may be beneficial forpatients to alternate or cycle between multiple different treatments, toreduce the risk of their developing inhibitory antibodies. Thus, abispecific antigen binding molecule of the present invention may beadministered to a patient who has previously received treatment with adifferent FVIIIa-activity replacing polypeptide drug, e.g., a bispecificantigen binding molecule for FIXa and FX, optionally emicizumab, evenwhere the patient has not (yet) developed inhibitory antibodies.Similarly, emicizumab or other bispecific antigen binding molecules forFIXa and FX, and other FVIIIa-activity replacing polypeptide drugsgenerally, may be administered to patients who were previously treatedwith a bispecific antigen binding molecule of the present invention.Regiments of treatment may comprise administration of a firstFVIII-activity replacing polypeptide drug for a first period (e.g.,between one and six months, or between six months and one year),followed by switching to a different FVIII-activity replacingpolypeptide drug for a second period (e.g. between one and six months,or between six months and one year), followed by switching back to thefirst drug or switching to yet another FVIII-activity replacingpolypeptide drug. The different amino acid sequences of the differentdrug treatments should ensure that a patient at risk of developinginhibitory antibodies to one drug is no longer at risk of developinginhibitory antibodies to the first drug (e.g., emicizumab) followingswitching to a different drug (e.g., a molecule of the presentinvention). The cycling period may be varied or shortened, according toconvenience and the preferences of the patient and doctor.

It will be recognised that administration of the encoding nucleic acidrepresents an alternative therapy, and may be performed in place ofadministering the polypeptide drug directly.

As noted, the bispecific antigen-binding molecules of the presentinvention are believed to have a strong safety profile, associated withno (or minimal) incidents of hypersensitivity reactions, development ofallo-antibodies, organ toxicity or other adverse events leading todiscontinuation of the therapy.

Clauses

The following numbered clauses represent embodiments of the inventionand are part of the description.

1. Bispecific antibody that binds FIXa and FX and catalysesFIXa-mediated activation of FX, wherein the antibody comprises twoimmunoglobulin heavy-light chain pairs, wherein

-   -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain comprises a set of HCDRs comprising HCDR1,        HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1        is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is SEQ ID        NO: 408, and/or wherein the first VH domain is at least 95        identical to the N1280H VH domain at the amino acid sequence        level;    -   the second VH domain is at least 95% identical to the T0201H VH        domain SEQ ID NO: 470 at the amino acid sequence level, and    -   the first VL domain and the second VL domain each comprise a set        of LCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid        sequences defined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID        NO: 7 and LCDR3 is SEQ ID NO: 8, and/or wherein the first VL        domain and the second VL domain are at least 95% identical to        the 0128L VL domain SEQ ID NO: 10 at the amino acid sequence        level.        2. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain is a product of recombination of human        immunoglobulin heavy chain v, d and j gene segments, wherein the        v gene segment is IGHV3-7 (e.g., VH3-7*01) and the j gene        segment is IGHJ6 (e.g., JH6*02),    -   the second VH domain is a product of recombination of human        immunoglobulin heavy chain v, d and j gene segments, wherein the        v gene segment is IGHV1-46 (e.g., VH1-46*03) and the j gene        segment is IGHJ1 (e.g., JH1*01), and optionally wherein the d        gene segment is IGHD6-6 (e.g., DH6-6*01), and    -   the first VL domain and the second VL domain are both products        of recombination of human immunoglobulin light chain v and j        gene segments, wherein the v gene segment is IGLV3-21 (e.g.,        VL3-21*d01) and the j gene segment is IGLJ2 (e.g., JL2*01) or        IGLJ3 (e.g., JL3*02).        3. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein    -   the first VH domain has at least 95% amino acid sequence        identity with the N1280H VH domain SEQ ID NO: 443,    -   the second VH domain has at least 95% amino acid sequence        identity with the T0201H VH domain SEQ ID NO: 470, and    -   the first VL domain and the second VL domain each have at least        95% amino acid sequence identity with the 0128L VL domain SEQ ID        NO: 10.        4. Bispecific antibody according to any preceding clause,        wherein the first VH domain comprises a set of HCDRs comprising        HCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein        HCDR1 is SEQ ID NO: 406, HCDR2 is SEQ ID NO: 407 and HCDR3 is        SEQ ID NO: 408.        5. Bispecific antibody according to clause 4, wherein the first        VH domain comprises HCDR1 SEQ ID NO: 441.        6. Bispecific antibody according to clause 4 or clause 5,        wherein the first VH domain comprises HCDR2 SEQ ID NO: 634.        7. Bispecific antibody according to any of clauses 4 to 6,        wherein the first VH domain comprises HCDR2 SEQ ID NO: 436.        8. Bispecific antibody according to any of clauses 4 to 7,        wherein the first VH domain comprises HCDR3 SEQ ID NO: 635.        9. Bispecific antibody according to any of clauses 4 to 8,        wherein the first VH domain comprises HCDR3 SEQ ID NO: 433.        10. Bispecific antibody according to any preceding clause,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1280H.        11. Bispecific antibody according to any preceding clause,        wherein the the first VH domain comprises a set of N1280H HCDRs        comprising N1280H HCDR1 SEQ ID NO: 441, N1280H HCDR2 SEQ ID NO:        436 and N1280H HCDR3 SEQ ID NO: 433.        12. Bispecific antibody according to clause 10 or clause 11,        wherein the first VH domain is the N1280H VH domain SEQ ID NO:        443.        13. Bispecific antibody according to any of clauses 1 to 11,        wherein the first VH domain is the N1454H VH domain SEQ ID NO:        454.        14. Bispecific antibody according to any of clauses 1 to 11,        wherein the first VH domain is the N1441H VH domain SEQ ID NO:        456.        15. Bispecific antibody according to any of clauses 1 to 11,        wherein the first VH domain is the N1442H VH domain SEQ ID NO:        458.        16. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1333H.        17. Bispecific antibody according to clause 16, wherein the        first VH domain comprises a set of N1333H CDRs comprising N1333H        CDR1, N1333H CDR2 and N1333H CDR3.        18. Bispecific antibody according to clause 16 or clause 17,        wherein the first VH domain is the N1333H VH domain.        19. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1327H.        20. Bispecific antibody according to clause 19, wherein the the        first VH domain comprises a set of N1327H HCDRs comprising        N1327H HCDR1, N1327H HCDR2 and N1327H HCDR3.        21. Bispecific antibody according to clause 19 or clause 20,        wherein the first VH domain is the N1327H VH domain.        22. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1314H.        23. Bispecific antibody according to clause 22, wherein the the        first VH domain comprises a set of N1314H HCDRs comprising        N1314H HCDR1, N1314H HCDR2 and N1314H HCDR3.        24. Bispecific antibody according to clause 22 or clause 23,        wherein the first VH domain is the N1314H VH domain.        25. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1172H.        26. Bispecific antibody according to clause 25, wherein the the        first VH domain comprises a set of N1172H HCDRs comprising        N1172H HCDR1, N1172H HCDR2 and N1172H HCDR3.        27. Bispecific antibody according to clause 25 or clause 26,        wherein the first VH domain is the N1172H VH domain.        28. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N1091H.        29. Bispecific antibody according to clause 28, wherein the the        first VH domain comprises a set of N1091H HCDRs comprising        N1091H HCDR1, N1091H HCDR2 and N1091H HCDR3.        30. Bispecific antibody according to clause 28 or clause 29,        wherein the first VH domain is the N1091H VH domain.        31. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N0511H.        32. Bispecific antibody according to clause 31, wherein the the        first VH domain comprises a set of N0511H HCDRs comprising        N0511H HCDR1, N0511H HCDR2 and N0511H HCDR3.        33. Bispecific antibody according to clause 31 or clause 32,        wherein the first VH domain is the N0511H VH domain.        34. Bispecific antibody according to any of clauses 1 to 3,        wherein the first VH domain has at least 96%, at least 97%, at        least 98% or at least 99% amino acid sequence identity to        N0436H.        35. Bispecific antibody according to clause 34, wherein the the        first VH domain comprises a set of N0436H HCDRs comprising        N0436H HCDR1, N0436H HCDR2 and N0436H HCDR3.        36. Bispecific antibody according to clause 34 or clause 35,        wherein the first VH domain is the N0436H VH domain.        37. Bispecific antibody according to any preceding clause,        wherein the second VH domain has at least 95%, at least 96%, at        least 97%, at least 98% or at least 99% amino acid sequence        identity to T0201H VH domain SEQ ID NO: 470.        38. Bispecific antibody according to clause 37, wherein the        second VH domain comprises an HCDR1 which is the T0201H HCDR1        SEQ ID NO: 462, an HCDR2 which is the T0201H HCDR2 SEQ ID NO:        467, and/or an HCDR3 which is the T0201H HCDR3 SEQ ID NO: 468.        39. Bispecific antibody according to any preceding clause        wherein the second VH domain comprises HCDR1 SEQ ID NO: 636.        40. Bispecific antibody according to clause 39, wherein the        second VH domain comprises HCDR1 SEQ ID NO: 598.        41. Bispecific antibody according to any preceding clause,        wherein the second VH domain comprises HCDR2 SEQ ID NO: 467.        42. Bispecific antibody according to any preceding clause,        wherein the second VH domain comprises HCDR3 SEQ ID NO: 637.        43. Bispecific antibody according to clause 42, wherein the        second VH domain comprises HCDR3 SEQ ID NO: 638.        44. Bispecific antibody according to clause 43, wherein the        second VH domain comprises HCDR3 SEQ ID NO: 639.        45. Bispecific antibody according to clause 44, wherein the        second VH domain comprises HCDR3 SEQ ID NO: 565.        46. Bispecific antibody according to clause 44, wherein the        second VH domain comprises HCDR3 SEQ ID NO: 583.        47. Bispecific antibody according to any of clauses 37 to 45,        wherein the second VH domain comprises SEQ ID NO: 632.        48. Bispecific antibody according to any of clauses 37 to 45,        wherein the second VH domain comprises SEQ ID NO: 600.        49. Bispecific antibody according to any of clauses 37 to 46,        wherein the second VH domain comprises SEQ ID NO: 585.        50. Bispecific antibody according to clause 38, wherein the the        second VH domain comprises a set of T0201H HCDRs comprising        T0201H HCDR1, T0201H HCDR2 and T0201H HCDR3.        51. Bispecific antibody according to clause 37, clause 38 or        clause 50, wherein the second VH domain is the T0201H VH domain,        optionally with a substitution at Cys114.        52. Bispecific antibody according to clause 51, wherein the        substitution at Cys114 is Ile, Gln, Arg, Val or Trp.        53. Bispecific antibody according to any of clauses 1 to 38,        wherein the second VH domain comprises a set of T0638H HCDRs        comprising T0638H HCDR1, T0638H HCDR2 and T0638H HCDR3.        54. Bispecific antibody according to clause 53, wherein the        second VH domain is the T0638 VH domain, optionally with a        substitution at Cys114.        55. Bispecific antibody according to clause 54, wherein the        substitution at Cys114 is Ile, Gln,

Arg, Val or Trp.

56. Bispecific antibody according to any preceding clause, wherein thefirst VL domain and the second VL domain each have at least 96%, atleast 97%, at least 98% or at least 99 amino acid sequence identity with0128L SEQ ID NO: 10.

57. Bispecific antibody according to any preceding clause, wherein thefirst VL domain and the second VL domain each comprise a set of 0128LCDRs comprising 0128L LCDR1 SEQ ID NO: 6, 0128L LCDR2 SEQ ID NO: 7 and0128L LCDR3 SEQ ID NO: 8.

58. Bispecific antibody according to any preceding clause, wherein thefirst VL domain and the second VL domain are identical in amino acidsequence.

59. Bispecific antibody according to clause 58, wherein the first VLdomain and the second VL domain comprise the 0325L amino acid sequenceSEQ ID NO: 416.

60. Bispecific antibody according to clause 58 or clause 59, wherein thefirst VL domain and the second VL domain comprise the 0128L amino acidsequence SEQ ID NO: 10.

61. Bispecific antibody according to any preceding clause, wherein eachheavy-light chain pair further comprises a CL constant domain pairedwith a CH1 domain.

62. Bispecific antibody according to any preceding clause, wherein theheavy-light chain pairs comprise a common light chain.

63. Bispecific antibody according to clause 62, wherein the common lightchain comprises the CL amino acid sequence SEQ ID NO: 146 of the 0128Llight chain.

64. Bispecific antibody according to clause 63, wherein the common lightchain is the 0325L light chain SEQ ID NO: 414.

65. Bispecific antibody according to clause 63, wherein the common lightchain is the 0128L light chain SEQ ID NO: 405.

66. Bispecific antibody according to any preceding clause, wherein theheavy chain of each heavy-light chain comprises a heavy chain constantregion and wherein the first and second heavy-light chain pairsassociate to form tetrameric immunoglobulin through dimerisation of theheavy chain constant regions.67. Bispecific antibody according to clause 66, wherein the heavy chainconstant region of the first heavy-light chain pair comprises adifferent amino acid sequence from the heavy chain constant region ofthe second heavy-light chain pair, wherein the different amino acidsequences are engineered to promote heterodimerisation of the heavychain constant regions.68. Bispecific antibody according to clause 67, wherein the heavy chainconstant regions comprise knobs-into-holes mutations or charge pairmutations.69. Bispecific antibody according to clause 67, wherein the heavy chainconstant region of one (e.g., the first) heavy-light chain pair is ahuman IgG4 constant region comprising substitution K439E and wherein theheavy chain constant region of the other (e.g., the second) heavy-lightchain pair is an IgG4 region comprising substitution E356K, whereinconstant region numbering is according to the EU numbering system.70. Bispecific antibody according to any of clauses 66 to 69, whereinthe heavy chain constant region of one or both heavy-light chain pairsis a human IgG4 constant region comprising substitution S228P, whereinconstant region numbering is according to the EU numbering system.71. Bispecific antibody according to any of clauses 66 to 70, whereinthe heavy chain constant region of one (e.g., the first) heavy-lightchain pair comprises SEQ ID NO: 409 and the heavy chain constant regionof the other (e.g., the second) heavy-light chain pair comprises SEQ IDNO: 410.72. Bispecific antibody according to any of clauses 66 to 71, comprising

-   -   a first heavy chain comprising a first VH domain amino acid        sequence SEQ ID NO: 443 or SEQ ID NO: 456,    -   a second heavy chain comprising a second VH domain amino acid        sequence SEQ ID NO: 632, and    -   a common light chain comprising a VL domain amino acid sequence        SEQ ID NO: 416.        73. Bispecific antibody according to any of clauses 66 to 72,        comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        419,    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        421, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        74. Bispecific antibody according to any of clauses 66 to 71,        comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        424    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        421, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        75. Bispecific antibody according to any of clauses 66 to 72,        comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        426    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        421, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        76. Bispecific antibody according to any of clauses 66 to 71,        comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        428    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        430, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        77. Bispecific antibody according to any of clauses 66 to 71,        comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        428    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        432, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        78. Bispecific antibody according to clause 66, wherein the        heavy chain constant region of the first heavy-light chain pair        is identical to the heavy chain constant region of the second        heavy-light chain pair.        79. Bispecific antibody according to any of clauses 1 to 68,        wherein the antibody is human IgG.        80. Bispecific antibody according to clause 79, wherein the        antibody is human IgG4.        81. Bispecific antibody according to clause 79 or clause 80,        wherein the IgG comprises the IgG4-PE heavy chain constant        region SEQ ID NO: 143, optionally engineered with one or more        amino acid substitutions to promote heterodimerisation.        82. Bispecific antibody according to clause 79 or clause 80,        wherein the antibody comprises the Fc region of emicizumab.        83. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1280H VH domain SEQ ID NO: 443, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0999H VH domain SEQ ID NO: 632, and        wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0325L light chain amino acid        sequence SEQ ID NO: 414.        84. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1454H VH domain SEQ ID NO: 454, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0999H VH domain SEQ ID NO: 632, and        wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0325L light chain amino acid        sequence SEQ ID NO: 414.        85. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1441H VH domain SEQ ID NO: 456, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0999H VH domain SEQ ID NO: 632, and        wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0325L light chain amino acid        sequence SEQ ID NO: 414.        86. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1442H VH domain SEQ ID NO: 458, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0736H VH domain SEQ ID NO: 600, and        wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0325L light chain amino acid        sequence SEQ ID NO: 414.        87. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1442H VH domain SEQ ID NO: 458, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0687H VH domain SEQ ID NO: 585, and        wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0325L light chain amino acid        sequence SEQ ID NO: 414.        88. Bispecific antibody that binds FIXa and FX and catalyses        FIXa-mediated activation of FX, wherein the antibody comprises        two immunoglobulin heavy-light chain pairs, wherein    -   a first heavy-light chain pair comprises a FIXa binding Fv        region comprising a first VH domain paired with a first VL        domain, wherein the first VH domain is at least 98% identical in        amino acid sequence to the N1333H VH domain, and    -   a second heavy-light chain pair comprises a FX binding Fv region        comprising a second VH domain paired with a second VL domain,        wherein the second VH domain is at least 98 identical in amino        acid sequence to the T0638H VH domain, and wherein    -   the first and second heavy-light chain pairs each comprise a        common light chain comprising the 0128L light chain amino acid        sequence SEQ ID NO: 405.        89. Bispecific antibody according to any preceding clause, which        reduces the coagulation time of FVIII-deficient human blood        plasma to less than 40 seconds in an aPTT assay.        90. Bispecific antibody according to clause 89, which reduces        the coagulation time of FVIII-deficient human blood plasma to        less than 30 seconds in an aPTT assay.        91. Bispecific antibody according to clause 90, which reduces        the coagulation time of FVIII-deficient human blood plasma to        22-28 seconds in an aPTT assay.        92. Bispecific antibody according to clause 91, which reduces        the coagulation time of FVIII-deficient human blood plasma to        24-26 seconds in an aPTT assay.        93. Bispecific antibody according to any preceding clause, which        enhances the FIXa-mediated activation of FX to FXa to the same        or similar extent as emicizumab in a FXase assay.        94. Bispecific antibody according to any preceding clause, which        enhances the FIXa-mediated activation of FX to FXa to at least        the same extent as emicizumab.        95. Bispecific antibody according to any of clauses 89 to 94,        wherein said coagulation time or FIXa-mediated activation is as        determined at an antibody concentration of 0.1 mg/ml, 0.3 mg/ml        or 0.5 mg/ml at 37 degrees C.        96. Bispecific antibody according to any preceding clause,        wherein the antibody has an EC50 for Cmax in a fluorometric        thrombin generation assay (TGA) that is within 10% of or is        lower than the Cmax EC50 of emicizumab in said assay, and/or        wherein the antibody generates a maximal response of Cmax        between 200 and 450 nM thrombin in a fluorometric TGA.        98. Bispecific antibody according to clause 96, wherein the        antibody has an EC50 of less than 50 nM for Cmax in a        fluorometric TGA.        99. Bispecific antibody according to clause 98, which has an        EC50 of less than 10 nM for Cmax in a fluorimetric TGA.        100. Bispecific antibody according to any preceding clause,        wherein the maximal response of Cmax is between 250 nM and 400        nM.        101. Bispecific antibody according to any preceding clause,        wherein the antibody has an EC50 for Tmax in a fluorimetric TGA        that is within 10% of, or is lower than, the Tmax EC50 of        emicizumab in said assay, and/or wherein the antibody generates        a maximal response of Tmax between 1 and 10 minutes.        102. Bispecific antibody according to any preceding clause,        wherein the antibody has an EC50 of less than 5 nM for Tmax in a        fluorimetric TGA.        103. Bispecific antibody according to clause 102, wherein the        EC50 for Tmax is less than 2 nM.        104. Bispecific antibody according to any preceding clause,        wherein the antibody generates a maximal response of Tmax        between 2 and 8 minutes in a fluorimetric TGA.        105. Anti-FIXa antibody comprising two copies of the first        heavy-light chain pair as defined in any preceding clause.        106. Anti-FX antibody comprising two copies of the second        heavy-light chain pair as defined in any of clauses 1 to 104.        107. Isolated nucleic acid encoding an antibody according to any        preceding clause.        108. A host cell in vitro comprising recombinant DNA encoding an        antibody heavy chain comprising a first VH domain as defined in        any of clauses 1 to 104,    -   an antibody heavy chain comprising a second VH domain as defined        in any of clauses 1 to 104, and/or    -   an antibody light chain comprising a first or second VL domain        as defined in any of clauses 1 to 104.        109. A host cell according to clause 108 comprising recombinant        DNA encoding    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        419 or SEQ ID NO: 426,    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        421, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414.        110. A population of host cells in vitro, wherein each host cell        comprises recombinant DNA encoding a bispecific antibody        according to any of clauses 1 to 104.        111. A kit for production of a bispecific antibody according to        any of clauses 1 to 104, comprising    -   an antibody heavy chain comprising a first VH domain as defined        in any of clauses 1 to 104, or nucleic acid encoding said heavy        chain,    -   an antibody heavy chain comprising a second VH domain as defined        in any of clauses 1 to 104, or nucleic acid encoding said heavy        chain,    -   an antibody light chain comprising a first VL domain as defined        in any of clauses 1 to 104, or nucleic acid encoding said light        chain, and    -   an antibody light chain comprising a second VL domain as defined        in any of clauses 1 to 104, or nucleic acid encoding said light        chain.        112. A kit according to clause 111, comprising    -   a first heavy chain comprising amino acid sequence SEQ ID NO:        419 or SEQ ID NO: 426, or nucleic acid encoding said first heavy        chain,    -   a second heavy chain comprising amino acid sequence SEQ ID NO:        421, or nucleic acid encoding said second heavy chain, and    -   a common light chain comprising amino acid sequence SEQ ID NO:        414, or nucleic acid encoding said common light chain.        113. A kit according to clause 111 or clause 112, wherein said        amino acid sequences or said nucleic acids are provided in        cells.        114. A kit according to clause 111 or clause 112, wherein said        amino acid sequences or said nucleic acids are provided in        cell-free buffered aqueous media.        115. A kit according to any of clauses 111 to 114, wherein each        of said amino acid sequences or each of said nucleic acids is        provided in a separate phial.        116. A method of producing a bispecific antibody according to        any of clauses 1 to 104, comprising culturing host cells        according to clause 108 or clause 109 under conditions for        expression of the bispecific antibody, and recovering the        bispecific antibody from the host cell culture.        117. A method according to clause 116, comprising culturing the        host cells in a vessel comprising a volume of at least 100        litres.        118. A method according to clause 117, wherein the vessel is of        stainless steel or is a single-use bioreactor.        119. A composition comprising a bispecific antibody according to        any of clauses 1 to 104, or isolated nucleic acid according to        clause 107, in solution with a pharmaceutically acceptable        excipient.        120. A composition according to clause 119, wherein the        bispecific antibody or nucleic acid is in sterile aqueous        solution.        121. A composition according to clause 119 or clause 120,        comprising a bispecific antibody according to any of clauses 1        to 104 wherein the bispecific antibody is at least 95% pure such        that the composition comprises no more than 5% homodimeric        antibody contaminants.        122. A composition according to clause 121, wherein the        bispecific antibody is at least 99 pure such that the        composition comprises no more than 1% homodimeric antibody        contaminants.        123. A method of controlling bleeding in a patient, comprising        administering a composition according to any of clauses 119 to        122 to the patient.        124. A composition according to any of clauses 119 to 122 for        use in a method of treatment of the human body by therapy.        125. A composition according to any of clauses 119 to 122 for        use in a method of controlling bleeding in a patient.        125. Use of a bispecific antibody according to any of clauses 1        to 104 for the manufacture of a medicament for controlling        bleeding in a haemophilia A patient.        126. A method according to clause 123, or a composition for use        or use according to clause        125, wherein the patient is a haemophilia A patient.        127. A method or a composition for use according to clause 126,        wherein the patient is resistant to treatment with FVIII owing        to the presence of inhibitory antibodies in the bloodstream.        128. A method or a composition for use according to clause 126        or clause 127, wherein the patient is resistant to treatment        with another bispecific antibody to FIXa and FX owing to the        presence of inhibitory antibodies in the bloodstream.        129. A method or a composition for use according to clause 128,        wherein the patient is resistant to treatment with emicizumab.        130. A method of reducing development of inhibitory anti-drug        antibodies in a haemophilia A patient undergoing treatment with        a polypeptide that replaces FVIIIa activity, comprising    -   administering a first FVIIIa-activity replacing polypeptide drug        to the patient for a period of 1-12 months,    -   switching the patient to a second, different FVIIIa-activity        replacing polypeptide drug for a period of 1-12 months, and    -   switching the patient to either the first antigen-binding        molecule or to a third, different FVIIIa-activity replacing        polypeptide drug for a period of 1-12 months, wherein    -   the first, second or third FVIIIa-activity replacing polypeptide        drug is a bispecific antibody according to any of clauses 1 to        104,    -   and wherein in each case the FVIIIa-activity replacing        polypeptide drug or its encoding nucleic acid is administered in        a therapeutically effective amount to functionally replace        FVIIIa in the patient, and wherein the risk of the patient        developing inhibitory anti-drug antibodies to any of the        FVIIIa-activity replacing polypeptide drug is reduced compared        with a patient continuing to receive treatment with that        FVIIIa-activity replacing polypeptide drug.        131. A composition comprising a FVIIIa-activity replacing        polypeptide drug or its encoding nucleic acid, for use in a        method of treating a haemophilia A patient while reducing        development of inhibitory anti-drug antibodies, or use of a        FVIIIa-activity replacing polypeptide drug or its encoding        nucleic acid for the manufacture of a medicament for use in a        method of treating a haemophilia A patient while reducing        development of inhibitory anti-drug antibodies, the method        comprising    -   administering a first FVIIIa-activity replacing polypeptide drug        to the patient for a period of 1-12 months,    -   switching the patient to a second, different FVIIIa-activity        replacing polypeptide drug for a period of 1-12 months, and    -   switching the patient to either the first antigen-binding        molecule or to a third, different FVIIIa-activity replacing        polypeptide drug for a period of 1-12 months, wherein    -   the first, second or third FVIIIa-activity replacing polypeptide        drug is a bispecific antibody according to any of clauses 1 to        104,    -   and wherein in each case the FVIIIa-activity replacing        polypeptide drug or its encoding nucleic acid is administered in        a therapeutically effective amount to functionally replace        FVIIIa in the patient, and wherein the risk of the patient        developing inhibitory anti-drug antibodies to any of the        FVIIIa-activity replacing polypeptide drug is reduced compared        with a patient continuing to receive treatment with that        FVIIIa-activity replacing polypeptide drug.        132. A method according to clause 130, or a composition for use        or use according to clause        131, wherein the first, second and third FVIIIa-activity        replacing polypeptide drugs are recombinant or plasma-derived        FVIII, emicizumab, and a bispecific antibody according to any of        clauses 1 to 104, in any order.        133. A method, composition for use or use according to any of        clauses 123 to 132 wherein the treatment comprises subcutaneous        administration of the composition to the patient.

Equivalents: Those skilled in the art will recognise, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be within the scope of protection of theappended claims.

EXAMPLES

Bispecific IgG antibodies comprising Fv binding sites for human FIXa andhuman FX were generated as described in PCT/EP2018/066836 filed on 22Jun. 2018 entitled “Bispecific antibodies for factor IX and factor X”(WO2018/234575). As described therein, an extensive campaign ofimmunisation and screening led to the identification of an anti-FIXaantibody NINA-0128 which, when paired in IgG format with any of aselection of different anti-FX binding Fvs, showed outstanding activityin functional screens including a tenase assay and aPTT assay. NINA-0128comprises VH domain N0128H and VL domain 0128L. A number of variants ofthe N0128H VH domain were generated and tested, resulting in furtherimprovements in function in a bispecific format, including for examplethe N0436H VH domain.

Building on that work, bispecific IgG were designed with the VL domainof NINA-0128 as a common light chain. A panel of anti-FX antibodies weregenerated in vivo in a transgenic mouse comprising human immunoglobulingenes. These were co-expressed with NINA-0128 as the anti-FIX bindingarm, using the 0128L VL domain in a common light chain including a humanconstant region. One VH domain, T0200, showed outstanding activity inthe bispecific format and was selected for further development.Structurally related antibodies obtained from the same immunised animalas the T0200H clone, included further anti-FX VH domains that performedeven better than the T0200H VH domain in bispecific IgG4 with ananti-FIX VH domain and the 0128L common light chain.

Meanwhile, further anti-FIXa antibody variants were generated,introducing mutations in the VH domain while retaining the common 0128LVL domain. The anti-FIXa N0436H VH domain sequence was optimised bysubstituting all possible amino acids at each position in CDR1, CDR2 andCDR3, expressing the resulting VH domain variants in the context ofbispecific antibodies comprising the common light chain, evaluating thevariant bispecific antibodies in a range of functional assays,identifying mutations associated with increased functional activity, andgenerating further variants including combinations of mutationsassociated with increased functional activity.

Improved T0200H VH domain variants were combined with improved N0436H VHdomain variants, each paired with the N0128L common light chain, andrepeated rounds of optimisation, screening and selection were conducted.

FIG. 6 shows a simplified overview of the screening program.

Very strong FVIII mimetic activity was achieved with common light chainbispecific antibodies including the optimised sequences. The followingbispecific antibodies are examples of strong performers, as indicated byfunctional characterisation in a range of disease-relevant assays.Nomenclature of the bispecific antibodies which have a common lightchain is IXAX-nnnn.tttt.llll, wherein nnnn is a 4 digit numericalidentifier of the anti-FIX VH domain, tttt is a 4 digit identifier ofthe anti-FX VH domain, and 1111 is a 4 digit numerical identifier of thecommon VL domain:

-   -   IXAX-0128.0201.0128 (anti-FIXa VH domain N0128H; anti-FX VH        domain T0201H; 0128L common light chain)    -   IXAX-0436.0201.0128    -   IXAX-0511.0201.0128    -   IXAX-1091.0201.0128    -   IXAX-1172.0201.0128    -   IXAX-1280.0201.0128    -   IXAX-1341.0201.0128    -   IXAX-1327.0201.0128    -   IXAX-1333.0201.0128

Other high performing anti-FX VH domains which combine well with theabove and other anti-FIX VH domains in bispecific antibodies were thoseof the T0201H lineage and variants thereof such as those listed in FIG.11 and other related sequences. Particularly good results were obtainedwith bispecific antibodies including an anti-FIX VH domain, an anti-FXVH domain and a common light chain selected from the following:

anti-FIX VH domain: anti-FX VH domain: common VL domain: N0436H T0201H0128L N0511H T0596H 0325L N1091H T0616H N1172H T0638H N1280H T0666HN1327H T0678H N1333H T0681H N1341H T0687H N1441H T0736H N1442H T0999HN1454H

For example,

-   -   IXAX-1280.0999.0325    -   IXAX-1454.0999.0325    -   IXAX-1441.0999.0325    -   IXAX-1441.0736.0325    -   IXAX-1442.0687.0325

The bispecific antibodies described here represent candidatepharmaceutical drug molecules for therapeutic use as described herein.They may offer a vital healthcare option for patients by providing analternative to existing treatments such as emicizumab, especially inpatients for whom such existing treatments are no longer effective dueto the presence of anti-drug antibodies.

In these Examples, the reference antibody AbE or Antibody E is abispecific antibody having the heavy and light chain amino acidsequences of emicizumab [3].

Example 1. Creation of Anti-FX Antibody Panel with Common Light Chain

Transgenic mice expressing a common light chain comprising the 0128L VLdomain of were immunised with human factor X. Antigen specific B cellswere single cell sorted by flow cytometry and the VH and VL sequenceswere retrieved by next generation sequencing (NGS). 200 anti-FX heavychains were identified by NGS analysis of the single cell sortedlymphocytes. Further bulk NGS analysis was performed on bone marrow andlymph node tissues harvested from the same transgenic animals.

Example 2. Creation of Anti-FIXaxFIX Bispecific Antibodies with CommonLight Chain

Each anti-FX heavy chain was expressed in HEK293 cells as bispecificantibody comprising the anti-FIX N0128H heavy chain and the 0128L commonlight chain. The bispecific antibodies were purified by Protein Asubstantially as described in PCT/EP2018/066836 filed on 22 Jun. 2018entitled “Bispecific antibodies for factor IX and factor X”, which isincorporated by reference herein.

Example 3. Initial Screening of Anti-FX Arms Using ActivationCoagulation Factor VIII (FVIIIa)-Like Activity Assay (FXase or TenaseAssay)

200 bispecific antibodies comprising a range of different anti-FX heavychains, each in combination with the N0128H anti-FIX heavy chain and0128L common VL domain, were screened using a factor Xa generationassay. This functional screening detects FVIIIa-mimetic activity, i.e.,ability to enhance (catalyse) the FIXa-mediated activation of FX to FXa,in vitro by enzymatic “FXase” assay. In this assay, the test bispecificmolecule is contacted with FIXa and FX in the presence of phospholipid,under conditions suitable for formation of FXa. A substrate for FXa isadded which, when cleaved by FXa, generates a detectable product.Detection of this product in the presence of test bispecific antibody iscompared with a negative control in which no test antibody is present (acontrol antibody may be included). The detected signal is quantified byrecording absorbance of the reaction solution at 405 nm. Absorbance ismeasured across a range of antibody concentrations in the assay and anEC50 value is calculated as a measure of the bispecific antibody potencyin this assay. Significant difference of EC50 between test antibody andcontrol indicates that the test antibody is able to enhanceFIXa-mediated activation of FX. FIG. 7.

Results

Among all the bispecific antibodies assayed, a single one showedoutstanding FXase activity: the N0128H anti-FIX heavy chain and T0200Hanti-FX heavy chain, paired with 0128L common VL domain, had markedlyhigher FXase activity than all others in the panel. FIG. 8.

Materials & Methods—Standard FXase Reaction Conditions

7.5 μL FIX (3.75 μg/mL) and 5 μL supernatant from the Expi293 cellsproducing the recombinant antibodies (Example 8) were added to each wellof an assay plate and incubated at room temperature for 1 hour. Amixture of 2.5 μL FXIa (10 ng/mL), 5 μL FX (50 ng/mL), 0.05 μLphospholipid (10 mg/mL) and 5 μL TBSB-S buffer was added to each well toinitiate enzymatic reaction (FIXa cleavage of FX to generate FXa), andincubated at 37° C. for 1 hour. After 60 minutes, the reaction wasterminated by adding 5 μL of 0.5 M EDTA. After adding 10 μL S2765substrate solution to each well, absorbance at 405 nm (referencewavelength 655 nm) was measured for 30 minutes (one reading per 10minutes). All reactions were performed at 37° C. unless otherwisestated.

TBSB:

Tris buffered saline containing 0.1% bovine serum albumin

To make 7.5 mL TBSB:

0.1 mL 7.5% BSA solution (Sigma)

7.4 mL 1×TBS solution (diluted from 20×TBS solution ThermoFisher)

TBSB-S:

TBSB containing 5 mM CaCl2) and 1 mM MgCl2

To make 100 mL TBSB-S:

99.4 mL TBSB

0.5 mL 1M CaCl2) (Sigma)

0.1 mL 1M MgCl2 (Sigma)

FXIa Stock Solution (10 μg/mL):

Add 10 mL TBSB-S to 0.1 mg FXIa (Enzyme Research Laboratories) to make10 μg/mL stock solution.

Dilute to 10 ng/mL (1:1,000) working solution before use.

F.IXa Stock Solution (5 μg/mL)

Add 100 mL TBSB-S to 0.5 mg FIXa (HFIXa 1080) (Enzyme ResearchLaboratories) to make 5 μg/mL stock solution.

Dilute to 1.0 μg/mL (1:5) working solution before use.

FIX Stock Solution (37.5 μg/mL):

Add 13.3 mL TBSB-S to 0.5 mg FIX (Enzyme Research Laboratories) to make37.5 μg/mL stock solution.

Dilute to 3.75 μg/mL (1:10) working solution before use.

FX Working Solution (50 μg/mL):

Add 16 mL TBSB-S to 0.8 mg FX (Enzyme Research Laboratories) to make 50μg/mL working solution.

No further dilution is needed before use.

S2765 Stock Solution:

25 mg S2765 (Chromogenix) chromogenic substrate (0.035 mmol)

To make 2 mM stock solution:

Add 17.493 mL water to the vial and dissolve with shaking.

Polybrene Solution:

To make 0.6 g/L hexadimethrine bromide stock solution:

Add 0.15 g hexadimethrine bromide (Sigma) to 250 mL water.

Dilute to 0.6 mg/L (1:1,000) working solution before use.

S2765 Substrate Working Solution

A 1:1 mixture of 2 mM S-2765 stock solution and 0.6 mg/L polybrenesolution.

Example 4. Identification of Anti-FX T0200H VH Domain

The bispecific antibody designated IXAX.0128.0200.0128, comprisingN0128H anti-FIX VH domain, T0200H anti-FX VH domain and 0128L commonlight chain, demonstrated high FXase activity compared with the otherbispecific antibodies. The T0200H VH domain was chosen for furtherdevelopment to attempt production of yet further improved bispecificantibodies.

Example 5. Optimisation of Anti-FX T0200H VH Domain

Phylogenetic Analysis

From the bulk NGS (Example 1) and phylogenetic analysis, 113 anti-FXheavy chains were identified as belonging to the same lymphocyte clusteras the anti-FX heavy chain T0200H. The cluster represents B cells thatappear to share a common evolutionary lineage. The anti-FX heavy chainswithin the cluster shared approximately 95% sequence identity withT0200H at the amino acid level. FIG. 9.

The 113 anti-FX heavy chains were expressed in bispecific antibodieswith a panel of different anti-FIX heavy chains and the 0128L commonlight chain, and screened by FXase assay.

We identified several anti-FX-heavy chains that showed increased FXaseactivity compared with the T0200H VH domain when assayed as bispecificantibodies. FIG. 10 shows example data from the Xase assay.

A selection of the most active FX arm sequences is shown in FIG. 11.These VH domains were designated T0201H to T0217H respectively. Thosewith the strongest activity in bispecific format were T0204, T0207,T0205 and T0201 (FIG. 10b ).

Using amino acid sequence comparisons, and supported by the functionaldata, we identified several amino acid residues in frameworks and CDRregions of the anti-FX heavy chains that differ in the most active VHdomains and may contribute to the enhanced biological activity comparedwith T0200H. For example, one or more of the following amino acidfeatures of the VH domain may increase the FVIII-mimetic activity ofbispecific antibodies containing the VH domain (IMGT residue numbering):

-   -   Replacement of valine (V) by isoleucine (I) at position 5 in        FR1;    -   Replacement of lysine (K) by glutamine (Q) at position 13 in        FR1;    -   Replacement of leucine (L) by methionine (M) at position 39 in        FR2;    -   Replacement of threonine (T) by serine (S) at position 62 in        CDR2;    -   Replacement of aspartate (D) by serine (S) at position 64 in        CDR2;    -   Replacement of threonine (T) by serine (S) at position 85 in        FR3;    -   Replacement of alanine (A) by serine (S) at position 112 in the        CDR3.

Nevertheless it is clear that activity is high even without these aminoacid substitutions, since T0200H itself shows strong activity in abispecific antibody, and none of these substitutions was consistentlypresent in all of the top VH domains (FIG. 11).

Targeted Mutagenesis for Functional Optimisation

The CDR3 of VH domain T0201H was systematically mutated to provide alibrary of VH domains in which the residue at each position wasindividually replaced by another amino acid. The resulting VH domainswere named TOXXXH, where XXX numbers are shown in FIG. 12 for themutants of IMGT positions 114 (Cys), 115 (Leu), 116 (Gln) and 117 (Leu).Refer to FIG. 13 for IMGT numbering.

Removal of Potential Developmental Liability

An unpaired cysteine (C) residue present in CDR3 was identified as ahigh-risk sequence motif. This unpaired cysteine, present at position114 in the CDR3 of T0200H and all 113 further anti-FX VH domainsidentified from the bulk NGS analysis, represents a liability for thedevelopment of the bispecific antibody. We screened VH domainscontaining substitutions of all other amino acids for the cysteine atthis position in T0201H. These new variants were expressed with N1280H(see Example 6) and 0128L common light chain as IgG4 bispecificantibodies, purified by Protein A and screened for FVIII mimeticactivity by FXase assay. Replacement of cysteine at position 114 withisoleucine (I), glutamine (Q), arginine (R), valine (V) or tryptophan(VV) resulted in bispecifics antibodies with FVIII mimetic activitysimilar to bispecific antibodies having the T0201H or T0202H VH domains.We conclude C114 can be replaced with a variety of other amino acids andstill maintain FVIII mimetic activity.

Example 6. Systematic Sequence Optimisation of Anti-FIX VH

Each amino acid residue in CDR1, CDR2 and CDR3 was individually mutatedto generate single position mutants of the anti-FIX N0128H heavy chain.The anti-FIX heavy chain variants thus generated were expressed inbispecific format, paired with anti-FX heavy chain T0201H and N0128Lcommon VL domain in HEK293.

Protein A purified bispecific antibodies were assayed for biologicalactivity by FXase (Example 7) and aPTT to look for amino acid changesthat improved the FVIII-mimetic activity of the bispecific antibody.Improved variants were then combined to generate double or triplemutants in the CDR1, CDR2 and CDR3 regions.

Table N identifies mutants of the N0128H VH domain in which one or moreresidues of the CDRs are mutated to other amino acids. For example theN0436H VH domain is a Ser→Ile mutant of the N0128H VH domain, i.e., inwhich the serine at IMGT position 111A in CDR3 is replaced byisoleucine. Further residue mutations were introduced on top of initialsingle mutations. For example the N0511H VH domain is a Ser112ALysmutant of the N0436H VH domain, i.e., in which the serine at IMGTposition 112A in CDR3 is replaced by lysine. N1172H is a Glu64Arg mutantof the N0511H VH domain, i.e., in which the glutamate at IMGT position64 in CDR2 is replaced by arginine. N1280H is a Thr29Arg mutant of theN1172H VH domain, i.e., in which the Thr at IMGT position 29 in CDR1 isreplaced by arginine. The other named VH domains can be identified fromTable N in the same manner.

Refer to FIG. 14 for IMGT numbering.

Example 7. Screening of Improved Bispecific Antibodies in FXase Assay

Anti-FX arms comprising the VH domain variants generated as described inExample 5 were combined with anti-FIX arms comprising the VH domainvariants generated as described in Example 6, each paired with the 0128Lcommon VL domain, to generate FIXAxFX bispecific antibodies, andscreened for functional activity in the tenase assay.

Results

Example data are shown:

-   -   FIG. 10.    -   FIG. 15.    -   FIG. 16.

Highly active bispecific antibodies were identified for severalcombinations of anti-FIX VH and anti-FX VH domains, each paired with the0128L common VL domain. Examples of anti-FIX VH and anti-FX VH domaincombinations are shown in FIG. 16.

The identity of the anti-FX VH domain appeared to have a strongerinfluence than the identity of the anti-FIX VH domain for thesebispecific arm combinations, with T0638H, T0616H, T0596H and T0663Hbeing among the highest-performing anti-FX VH domains. These anti-FXdomains performed well in combination with a variety of anti-FIX arms,including variants of N1280H such as those indicated in FIG. 16.Anti-FIX VH domain sequences are identified by reference to appendedTable N. Anti-FX VH domain sequences are identified by reference to FIG.12.

FVIII-mimetic activity of N128 bispecific antibody was sequentiallyoptimised by modifying amino acid residues in any of the three CDRs.Several amino acid residues were identified to increase FVIII mimeticactivity across the CDRs and these mutations were combined to maximiseactivity. The FVIII mimetic activity of antibodies with the N0128H VHdomain was progressively improved with further VH domains in thefollowing order: N0128H→N0436H→N0511H→N1091H→N01172H→N1280H→N1333H. FIG.17.

Materials & Methods—Modified FXase Reaction Conditions.

Initial screening for bispecific antibody FVIII mimetic activity wasassessed using the Standard FXase Reaction Conditions set out above inExample 3. As the FVIII mimetic activity of the bispecific antibodyincreased, the Standard FXase reaction conditions were no longersufficient to detect improvements in FXase activity. Therefore, moresensitive Modified FXase Reaction Conditions were established.

This modified assay differs from the Standard FXase Reaction Conditionsin the following ways: FXIa is not used, the activated form of Factor IX(FIXa) is used and there is no incubation step. All FXase reagents aremixed with a bispecific antibody and the generation of FXa is detectedby recording the absorbance of the reaction solution 40 to 50 timesevery 30 seconds at 405 nm using an Envision plate reader set to 37° C.

18.45 μl TBSB-S buffer was mixed with 0.05 μl phospholipid (10 mg/ml)and mixed vigorously by pipetting to disperse the phospholipid. To thismixture 1.5 μl FIXa (1 μg/ml) and 5 μl of FX (50 μg/ml), was combinedwith 5 μl of polybrene (0.6 mg/L) and 5 μl S2765 (4 mM), all pre-warmedto 37° C. Finally, 5 ul of bispecific antibody being investigated forFXase activity was added. Absorbance at 405 nm (reference wavelength 655nm) was recorded 40 to 50 times every 30 seconds.

Example 8. Screening of Improved Bispecific Antibodies in PlasmaCoagulation Assay

Anti-FX arms comprising the VH domain variants generated as described inExample 5 were combined with anti-FIX arms comprising the VH domainvariants generated as described in Example 6, each paired with the 0128Lcommon VL domain, to generate FIXaxFX bispecific antibodies. Todetermine the ability of the bispecific antibodies of the presentinvention to correct the coagulation ability of the blood of haemophiliaA patients, the effect of these antibodies on the activated partialthromboplastin time (aPTT) using FVIII deficient plasma was examined.

A mixture of 5 μL of bispecific antibody solution having a variety ofconcentrations, 20 μL of FVIII deficient plasma (Helena Biosciences),and 25 μL of aPTT reagent (APTT Si L Minus, Helena Biosciences) waswarmed at 37° C. for 3 minutes. The coagulation reaction was initiatedby adding 25 μL of 25 mM CaCl₂ (Helena Biosciences) to the mixture. Thetime period until coagulation was measured. Apparatus used for this wasC-4 4 channel coagulation analyser (Helena Biosciences).

A sample of results is shown in FIG. 18.

Concentration dependency was subsequently determined for bispecificantibodies that exhibited the highest coagulation time-reducing effect.

For example, IXAX-1280.0201.0128 IgG4 antibody demonstrated a dosedependent decrease in aPTT, comparable to the reference antibody AbE(positive control). FIG. 19. No reduction in aPTT was observed for anisotype control antibody. Note that the antibody preparation used forthis assay was the result of a one-step purification on Protein A and assuch contained residual anti-FIX monospecific antibodies and residualanti-FX monospecific antibodies in addition to the desired bispecificfraction.

Example 9. Analysis of Anti-FIX VH Domains in Bispecific Antibodies

Considering data from a variety of functional assays including thosedescribed in Example 7 and Example 8, it was noted that the anti-FX VHdomain T0201 and its sequence variants performed well in combinationwith a variety of anti-FIX VH domains in bispecific antibodies with thecommon light chain. For example, anti-FIX VH domains N0128H, N0436H,N0511H, N1091H, N1172H, N1280H, N1314H, N1327H and N1333H all gave goodfunctional activity in the bispecific antibodies. These anti-FIX VHdomains share a close structural relationship. FIG. 20. Theirperformance could be further enhanced by fine tuning of residues throughsubstitution (Table N) and combining substitutions associated withimproved activity.

Example 10. Affinity for Antigen-Binding

Binding affinity and the kinetics of antibody-antigen interaction weredetermined using SPR. Affinity and kinetics of purified test antibodies(all IgG4PE) were compared to comparator anti-FIX antibody AbN orcomparator anti-FX antibody AbT as positive control and to an isotypecontrol (ISTC) as negative control.

Binding Affinity for FIX

The anti-FIX antibodies analysed showed binding to FIX in the affinityrange of approximately 0.18 μM to 0.3 μM and fast association (k_(on))and dissociation (k_(off)) rates for FIX. The anti-FIX antibodiesanalysed showed slightly higher binding affinity to FIX and higherassociation rate compared to the comparator antibody AbN. No binding toFIX was observed with ISTC. Table E-10-1.

TABLE E-10-1 Binding affinity and kinetic constants on-rate (kon) andoff- rate (koff) of anti-FIX antibodies. Anti-FIXa monospecific antibodynomenclature: NINA-hhhh.llll, wherein hhhh is the numeric identifier ofthe VH domain (e.g., N0436H) and llll is the numeric identifier of theVL domain (e.g., 0128L), Captured anti-FIX antibody k_(on) (1/Ms)k_(off) (1/s) K_(D) (M) NINA-0128 (n = 2 average) 2.92 × 10⁵ 5.76 × 10⁻²1.98 × 10⁻⁷ NINA-0436.0128 2.46 × 10⁵ 4.53 × 10⁻² 1.84 × 10⁻⁷NINA-0438.0128 2.30 × 10⁵ 6.73 × 10⁻² 2.93 × 10⁻⁷ NINA-0440.0128 1.85 ×10⁵ 5.35 × 10⁻² 2.89 × 10⁻⁷ NINA-0442.0128 1.94 × 10⁵ 4.71 × 10⁻² 2.42 ×10⁻⁷ NINA-0444.0128 2.16 × 10⁵ 4.45 × 10⁻² 2.06 × 10⁻⁷ NINA-0445.01282.04 × 10⁵ 5.44 × 10⁻² 2.67 × 10⁻⁷ NINA-0456.0128 1.51 × 10⁵ 3.96 × 10⁻²2.63 × 10⁻⁷ NINA-0460.0128 1.75 × 10⁵ 3.18 × 10⁻² 1.81 × 10⁻⁷ AbN 3.06 ×10⁴ 4.26 × 10⁻² 1.39 × 10⁻⁶ ISTC No binding No binding No bindingBinding Affinity for FX

The anti-FX antibodies analysed showed binding to FX in the affinityrange of approximately 0.1 μM to 1.4 μM and fast association (k_(on))and dissociation (_(koff)) rate for FX. No binding to FX was observedwith ISTC.

The anti-FX antibodies analysed similar binding affinity to FX comparedto the benchmark antibody AbT.

TABLE E-10-2 Binding affinity and kinetic constants on-rate (kon) andoff- rate (koff) of anti-FX antibodies. Anti-FX monospecific antibodynomenclature: TINA-hhhh.llll, wherein hhhh is the numeric identifier ofthe VH domain (e.g., N0201H) and llll is the numeric identifier of theVL domain (e.g., 0128L). Captured anti-FX antibody k_(on) k_(off) K_(D)(IgG4PE) (1/Ms) (1/s) (M) TINA-0200.0128 (n = 2 1.03 × 10⁵ 1.15 × 10⁻¹1.13 × 10⁻⁶ average) TINA-0215.0128 6.84 × 10⁴ 7.87 × 10⁻² 1.15 × 10⁻⁶TINA-0211.0128 6.57 × 10⁴ 4.47 × 10⁻² 6.80 × 10⁻⁷ TINA-0210.0128 7.64 ×10⁴ 7.44 × 10⁻² 9.74 × 10⁻⁷ TINA-0203.0128 6.80 × 10⁴ 5.98 × 10⁻² 8.80 ×10⁻⁷ TINA-0206.0128 6.72 × 10⁴ 6.67 × 10⁻² 9.92 × 10⁻⁷ TINA-0205.01286.26 × 10⁴ 8.53 × 10⁻² 1.36 × 10⁻⁷ TINA-0219.0128 1.01 × 10⁵ 9.11 × 10⁻²9.05 × 10⁻⁷ TINA-0217.0128 1.02 × 10⁵ 5.74 × 10⁻² 5.64 × 10⁻⁷TINA-0209.0128 5.90 × 10⁴ 6.79 × 10⁻² 1.15 × 10⁻⁶ TINA-0204.0128 1.09 ×10⁵ 7.85 × 10⁻² 7.18 × 10⁻⁷ TINA-0220.0128 5.38 × 10⁴ 5.60 × 10⁻² 1.04 ×10⁻⁶ TINA-0201.0128 8.67 × 10⁴ 5.02 × 10⁻² 5.79 × 10⁻⁷ TINA-0202.01288.87 × 10⁴ 7.20 × 10⁻² 8.12 × 10⁻⁷ TINA-0213.0128 9.69 × 10⁴ 1.33 × 10⁻¹1.37 × 10⁻⁶ TINA-0207.0128 1.66 × 10⁵ 1.41 × 10⁻¹ 8.47 × 10⁻⁷TINA-0214.0128 1.20 × 10⁵ 5.58 × 10⁻² 4.66 × 10⁻⁷ AbN No binding Nobinding No binding AbT 4.13 × 10⁴ 2.72 × 10⁻² 6.60 × 10⁻⁷ hIgG4PE ISTCNo binding No binding No bindingMaterials & Methods

SPR was used to determine the binding affinity (K_(D)) to FIX or FXrespectively, the kinetic constants on-rate (k_(on)) and off-rate(k_(off)). Analyses was performed using a Biacore 8K (GE Healthcare)system.

Anti-human IgG Fc antibody was immobilised on CM4 chip (GE Healthcare)according to the manufacturer's instructions. The chip surface wasactivated by amine coupling and subsequently blocked with 1Methanolamine. The immobilisation run was performed at 25° C. usingHBS-EP as immobilisation running buffer.

Monospecific antibodies (referred as ligand) which had been purified onProtein A were captured onto the anti-human IgG Fc CM4 surface atapproximately 2 μg/ml. The ligands were injected for 60 seconds at 10μl/min in all the active channels of all 8 flow channels. The run wasperformed at 25° C. using neutral pH HBS-P 1×+CaCl₂ 2.5 mM as runningbuffer.

Human FIX (MW ˜55 KDa) or human FX (MW ˜58 KDa) was reconstituted at 1mg/ml in the running buffer and used as analyte. The analyte wasinjected in multiple cycle kinetics (MCK) mode at 3 concentrations (1.5μM, 500 nM and 166.7 nM) with 120 seconds association phase and 200seconds (for FIX) or 300 seconds (for FX) dissociation phase, at flowrate 30 μl/sec in both active and reference channels. Three injectionsof 10 mM Glycine pH 1.5 for 60 sec. at 10 μl/min were used for theregeneration phase.

For the anti-FIX analysis, ISTC antibody hIgG4PE was captured at 1 μg/mlfor 60 seconds at 10 μl/min in the reference channel. hIgG4PE ISTC andhIgG1 ISTC were also captured in the active channel as a negativecontrol. The monospecific antibody AbN was used as positive control.

For the anti-FX analysis, the hIgG4PE ISTC was also captured in theactive channel as a negative control. The monospecific antibody AbT wasused as positive control.

The values for association rate constant (kon), dissociation rateconstant (koff) and dissociation constant (KD) were calculated from thebinding data by BIAevaluation software. Data were reference and buffersubtracted and fitted into one step biomolecular reaction (Langmuir 1:1)model. The first 30 seconds of dissociation were evaluated in the model.

Example 11. Simultaneous Binding of Bispecific Antibody to FX and FIX

The ability of FIXxFX bispecific antibody IXAX-0436.0202.0128 to bindsimultaneously to FIX and FX was demonstrated using SPR. The bindingkinetics of the purified bispecific antibody was compared to an isotypecontrol (ISTC). Sensorgrams of the binding indicated that the bispecificantibody bound simultaneously to FIX and FX while no binding to FIX andFX was observed with ISTC. FIG. 21A.

FIX was flown over the surface captured with the bispecific antibody toallow the binding with the first analyte. The interaction between thebispecific antibody generated a baseline response as indicated in thesensogram FIG. 21. The following injection of the second analyte, FX,generated a further increase in signal indicating that FX binds thebispecific antibody already in complex with FIX.

Contrarily no binding to FIX or FX was observed when FIX and FX wereflown over the surface where an isotype control was captured,demonstrating the specificity of interaction between FI and FX to thebispecific antibody. FIG. 21A.

Sensorgram for the bispecific antibody can also be compared withsensorgram for monospecific antibody. When the antibody captured is ananti-FX monospecific the same series of injection does not give anysignificant response when FIX is flown over instead when the secondinjection is performed (1:1 mixture) approximately 50 response units(RU) are observed while with the bispecific the response is 25 RUhigher. FIG. 21B.

A key feature of the FVII-mimetic bispecific antibody is the ability tobind simultaneously FIX and FX, to promote the conversion of FX into FXaby FIXa. The binding observed represents a biophysical confirmation thatthe bispecific antibodies described herein can interact simultaneouslywith Factor IX and Factor IX, which is in agreement with the functionaldata described in the accompanying Examples.

Materials & Methods

SPR analysis was performed using a Biacore 8K (GE Healthcare) system.

An anti-human IgG Fc antibody was immobilised on CM4 chip (GEHealthcare) according to the manufacturer's instructions. The chipsurface was activated by amine coupling and subsequently blocked with 1Methanolamine. The immobilisation run was performed at 25° C. usingHBS-EP as immobilisation running buffer.

Bispecific antibody (ligand), which had been purified by Protein Acapture followed by ion exchange chromatography, was captured on to theanti-human IgG Fc CM4 surface at approximately 2 μg/ml. The ligand wasinjected at 10 μg/ml for 60 seconds at 10 μl/min in one active channel.The run was performed at 25° C. using neutral pH HBS-P 1×+CaCl₂ 2.5 mMas running buffer.

Human FIX and human FX (analytes) were reconstituted at 1.15 mg/ml inthe running buffer and used as analytes. Analytes were injected at 10 μMalone or mixed 1:1 (10 μM 10 μM) at 10 μl/min for 180 seconds.

An isotype control hIgG4PE antibody was captured at 10 μg/ml for 60seconds at 10 μl/min in the reference channel as negative control. Ablank injection of buffer was performed for all the samples to be usedin the double referencing process. Three injections of 10 mM glycine pH1.5 for 30 seconds at 30 μl/min were used for the regeneration phase.The data were referenced and buffer subtracted and fitted into Langmuir1:1 model.

Example 12. Further Optimisation of Anti-FX VH

The anti-FIX binding arm of the bispecific antibody was “fixed” as a VHdomain comprising the CDRs of N1280H and a VL domain comprising the CDRsof 0128L, while further refinements were made to the anti-FX VH domainto improve performance. 0128L was used as a common light chain.

Table T identifies mutants of the T0201H VH domain in which one or moreresidues of the CDRs are mutated to other amino acids. The table showsthe name given to each variant VH domain having the identified mutation.In each case, residues other than those indicated are left unchanged.For example, the T0616H VH domain is a Leu115Ile mutant of the T0201H VHdomain, i.e., in which the leucine (L) at IMGT position 115 in CDR3 isreplaced by isoleucine (I). Further residue changes were introduced tothe variants containing the single mutations in the T0201H VH domain,resulting in further variants representing combinations of differentmutations in the T0201H VH domain. For example, the T0687H VH domain isa Ser111APhe, Cys114Val, Leu115Ile mutant of the T0201H VH domain, i.e.,in which the serine at IMGT position 111A in CDR3 is replaced byphenylalanine (T0537H mutation), the cysteine at IMGT position 114 inCDR3 is replaced by valine (T0606H mutation), and the leucine at IMGTposition 115 is replaced by isoleucine (T0616H mutation). Sequences ofother named anti-FX VH domains can be identified from Table T in thesame manner. Refer to FIG. 13 for IMGT numbering.

Bispecific antibodies, purified by Protein A chromatography, were testedfor functional activity to look for improvement over the parentbispecific comprising T0201H VH domain.

Improved antibodies were identified in the FXase assay (using ModifiedFXase Reaction Conditions as detailed in Example 7) and aPTT assay(method as detailed in Example 8).

Mutagenesis of HCDR3 produced improvements in FVIII mimetic activity.HCDRs of VH domains demonstrating improved activity are indicated inFIG. 25. For example, each of the following substitutions andcombinations of substitutions in the T0201H VH domain CDR3 was found toimprove FVIII mimetic activity (name of resulting VH domain indicated inbrackets) (non-exhaustive list):

-   -   Gln116Met in CDR3 (T0638H VH);    -   Leu115Ile in CDR3 (T0616H VH);    -   Ser111APhe in CDR3 (T0537H VH);    -   Cys114Ile Leu115Ile (T0666H VH);    -   Ser111APhe Cys114Ile Leu115Ile (T0678H VH);    -   Ser111APhe Cys114Leu Leu115Ile (T0681H VH);    -   Ser111APhe Cys114Val Leu115Ile (T0687H).

Concluding the HCDR3 mutagenesis of T0201H, the VH domains T0687H,T0678H and T0681H demonstrated the strongest activity in the bispecificantibodies.

Functional activity of the bispecific antibodies was still furtherimproved through mutagenesis of HCDR1 and HCDR2 in the anti-FX arm.Starting with T0681H, each amino acid residue of CDR1 and CDR2 wassystematically replaced by all other possible amino acids, generatingthe VH domains numbered T0690H to T0993H identified in Table T.

aPTT and TGA analyses were also conducted to support functionalassessment of HCDR1 variants. The VH domains T0736 (S29K mutation),T0713, T0734, T0742, T0774 and T0785 showed improved activity comparedwith T0681H. Based on the functional analyses of HCDR1 variants ofT0681H, VH domain T0736H was selected as the top performer. As comparedwith T0201H, T0736H combines a Ser29Lys substitution in CDR1 with theSer111APhe Cys114Val and Leu115Ile substitutions in CDR3.

FXase, aPTT and TGA analyses were also conducted to support functionalassessment of HCDR2 variants. Based on the functional analyses of HCDR2variants of T0681H, the following VH domains were identified to haveimproved activity compared with T0681H: T0926H (S62K), T850H (156L),T0925H (S62L), T0951H (G63S), T0958H (S64D), T0989 (T65R) and T0990H(T65S).

Selected CDR1 and CDR2 variants were then combined with selected CDR3,generating further VH domain variants to investigate possible furtherimprovements in activity.

FXase assay data for high-performing antibodies are summarised in FIG.22 and FIG. 23. The aPTT assay data are summarised in FIG. 24.

Bispecific antibodies comprising the VH domains shown in FIGS. 22 and 23demonstrated improved or similar clotting times compared with bispecificantibodies comprising T0201H, with T0999H demonstrating the shortestclotting time in aPTT assay.

FXase activity and clotting times were comparable with the comparatorbispecific antibody AbE.

FIG. 25 identifies CDRs of VH domains which were progressively improvedfor FVIII mimetic activity during the mutagenesis process.

Example 13. Strong Activity of Bispecific Antibodies in a ThrombinGeneration Assay

The thrombin generation assay (TGA) detects the activation ofprothrombin to thrombin in blood plasma. As thrombin is generated itconverts a fluorogenic substrate into a fluorophore, which iscontinuously monitored by a plate reader. The TGA provides a robustmeasure of the ability of bispecific antibodies to substitute for FVIIIin the coagulation cascade in FVIII-deficient plasma, and kinetics ofthrombin generation in the TGA are believed to be highly reliable as anindicator of in vivo therapeutic performance of FVIII-mimetic drugs.

Results

To establish a suitable concentration for factor IXa as a TGA trigger,we initially performed TGAs with a fixed concentration of bispecificantibody whilst varying the concentration of FIXa present in the triggerreagent. We determined that a stock solution of 1 ml MP reagentcontaining 222 nM FIXa is sufficient to trigger thrombin generation fornormal pooled human plasma (final concentration, 0.33 nM FIXa) with aCmax of 418.11 nM thrombin, a Tmax of 7.67 minutes and a lagtime of 5.83minutes. FIG. 26. These values are comparable with the reference rangein healthy adults (see, e.g., Table 3 of ref [11]) and validate the useof FIXa as a TGA trigger.

Bispecific antibody VH domain T0201H and CDR1, CDR2 and CDR3combinatorial variants of T0201H were expressed with FIX N1280H arm andN0128 common light chain in HEK cells, purified by protein Achromatography and analysed at a final concentration of 133 nM and 80nM. The VH domain variants exhibited shortening lagtime, increasing Cmaxand shorter time to peak compared with T0201H, with T0999 demonstratingthe largest thrombin peak height and shortest time to peak at bothconcentrations analysed. Performance of at least IXAX-1280.0999.0128 wascomparable with that of AbE and of the emicizumab calibrator. AbEdemonstrated a lagtime, peak height and time to peak of 2.5 mins, 291.8nM and 6.0 minutes respectively, and IXAX-1280.0999.0128 demonstrated alagtime, peak height and time to peak of 2.0 mins, 317.2 nM and 5minutes. FIG. 27. FIG. 28. As illustrated in FIG. 27 and in order ofincreasing Tmax, we observed a Tmax of 5, 6.83, 6.83, 7, 7.67, 13.17, 15and 15.67 minutes for bispecific antibodies comprising T0999, T0687,T0736, T0678, T0681, T0666, T0201 and T0596 respectively.

TABLE E13-1 Recorded parameters from TGA carried out on FVIII deficientplasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutantsof T0201H in bispecific antibody IXAX- 1280.0201.0128 at finalconcentration of 133 nM. ETP = endogenous thrombin potential. Isotype VHT0201 T0596 T0666 T0678 T0681 T0687 T0736 T0999 AbE Control Lagtime(min) 5.0 5.2 4.5 2.7 3.0 2.7 2.7 2.0 2.5 18.0 ETP (nmol/L 1774.4 1724.21883.6 2053.5 1932.4 2077.5 1805.4 2099.8 1951.9 −1.0 thrombin × min)Maximal Peak 134.9 126.4 160.4 259.6 235.6 267.8 237.5 317.2 291.8 19.9Height (nM) Time to peak 15.0 15.7 13.2 7.0 7.7 6.8 6.8 5.0 6.0 38.3(min) Velocity Index 13.5 12.0 18.5 59.9 50.5 64.4 57.1 105.7 83.6 1.0(nM/min) Tail Start (min) 39.0 39.8 37.0 29.8 30.2 29.5 28.2 26.7 27.3−1.0

TABLE E13-2 Recorded parameters from TGA carried out on FVIII deficientplasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutantsof T0201H in bispecific antibody IXAX-1280.0201.0128 at finalconcentration of 80 nM. ETP = endogenous thrombin potential. Refer toFIG. 27. Isotype VH T0201 T0596 T0666 T0678 T0681 T0687 T0736 T0999 AbEControl Lagtime (min) 5.0 5.3 4.7 2.7 3.0 2.7 2.7 2.0 3.0 19.0 ETP(nmol/L 1766.2 1793.8 1757.9 1975.4 1891.7 1927.1 1933.3 1986.1 1878.6−1.0 thrombin × min) Maximal Peak 126.6 125.9 148.2 269.7 231.4 266.2248.9 308.0 255.8 19.7 Height (nM) Time to peak 15.7 16.0 13.5 6.7 7.86.7 6.7 4.8 7.2 39.0 (min) Velocity Index 11.9 11.8 16.8 67.4 47.9 66.562.2 109.2 61.6 1.0 (nM/min) Tail Start (min) 41.0 41.5 37.0 28.7 29.829.5 30.0 26.8 29.8 −1.0

TABLE E13-2 Recorded parameters from TGA carried out with (i) emicizumabcalibrator and (ii) normal pooled plasma spiked with PBS. The emicizumabcalibrator is originally 100 ug/ml = 0.1 mg/ml = 666.666 nM. Dilution125/80 = 1.5625 (80 plasma, 20 Calibrator/MP, 20 FluCa, 5 PBS spike)provides final emicizumab concentration of 426.6 nM for the calibrator.Refer to FIG. 27. Calibrator Normal Plasma Lagtime (min) 2.0 4.8Endogenous Thrombin Potential 1538.0 1425.7 (ETP) (nmol/L thrombin ×min) Maximal Peak Height (nM) 308.6 371.2 Time to peak (min) 4.5 6.5Velocity Index (nM/min) 124.1 222.7 Tail Start (min) 23.7 22.7

For a dose response TGA, bispecific antibody IXAX-1280.0999.0128 wasexpressed in HEK cells, purified by Protein A chromatography andbispecific heterodimer purified by ion exchange chromatography. Using0.3 nM FIXa trigger, dose response of Cmax (nM) and Tmax (min) in theTGA was carried out on FVIII deficient plasma spiked with bispecificantibody IXAX-1280.0999.0128 and compared against emicizumab calibrator.Both bispecific antibodies demonstrated a linear decrease in Cmax withincreasing antibody concentration. IXAX-1280.0999.0128 achieved agreater Cmax than the calibrator antibody, this increase being morepronounced at lower bispecific antibody concentrations. See FIG. 29.

TABLE E13-3 Recorded parameters from TGA carried out on FVlll deficientplasma spiked with CDR1, CDR2 and CDR3 single and combinatorial mutantsof T0201H in bispecific antibody IXAX-1280.0201.0128. Refer to FIG. 29.IXAX-1280.0999.0128 Dose Response Flxa Trigger (0.3 nM) Normal Isotype300 nM 100 nM 30 nM 10 nM 3 nM 1 nM Plasma Control Lagtime (min) 2 1.832.17 3.33 4.5 5.83 6.17 18 Endogenous 2703 2170 2266 2311 2208 2088 1629−1 Thrombin Potential (ETP) (nmol/L thrombin × min) Maximal Peak 377.14348.38 327.7 274.76 208.45 124.11 466.31 23.47 Height (nM) Time to peak5.33 4.33 5.5 8 12 18.83 7.83 39.17 (min) Velocity Index 113.14 140.0198.31 58.88 27.82 9.62 279.78 1.11 (nM/min) Tail Start (min) 28.67 26.8328.67 31.5 35.33 44.5 23.33 −1

TABLE E13-4 Recorded parameters from TGA carried out with emicizumabcalibrator. Refer to FIG. 29 Emicizumab Calibrator Dose Response FlxaTrigger (0.3 nM) Normal Isotype 300 nM 100 nM 30 nM 10 nM 3 nM 1 nMPlasma Control Lagtime (min) 2 2.33 3 4.33 5.5 6.67 6.17 18 Endogenous1884 2363 2075 1733 1524 1634 1629 −1 Thrombin Potential (ETP) (nmol/Lthrombin × min) Maximal Peak 343.94 351.58 255.87 152.08 89.33 67.38466.31 23.47 Height (nM) Time to peak 4.17 5.5 8.17 12.5 18.33 23 7.8339.17 (min) Velocity Index 160.26 111.69 50.03 18.67 6.96 4.13 279.781.11 (nM/min) Tail Start (min) 25 27.67 29.33 35.33 43.33 58.17 23.33 −1Materials & Methods

For the initial experimental work to establish a suitable concentrationof factor IXa as a TGA trigger, 80 μl normal pooled plasma, taken fromhealthy individuals (Helena Biosciences), was mixed with 20 μl oftrigger reagent (Microparticle (MP) reagent which is composed ofphospholipids only containing varying amounts of FIXa) in Immulon 2HBtransparent U-bottom 96 well plates (ThermoFisher #3665). All reagentswere used according to manufacturers instructions, pre-warmed to 37° C.in a water bath.

Once a final concentration of 0.33 nM FIXa was determined to besufficient to trigger thrombin generation for normal plasma, the sameassay conditions including 0.3 nM FIXa were applied with FVIII-depletedplasma in calibrated automated thrombogram assays.

FVIII immunodepleted plasma (Helena Biosciences) was mixed with 20 μl oftrigger reagent (Microparticle (MP) reagent which is composed ofphospholipids only containing 222 nM FIXa, final concentration 0.33 nM)in Immulon 2HB transparent U-bottom 96 well plates. All reagents wereused according to manufacturers instructions, pre-warmed to 37° C. in awater bath. A TGA dose response was carried out starting at 300 nM oftest bispecific antibody or of emicizumab calibrator ((emicizumab spikedinto FVIII deficient plasma (Enzyme Research Laboratories)) with a 1 in3 dilution series over five points. A human IgG4 isotype controlantibody was used as negative control, and normal (FVIII+ve) pooledplasma spiked with PBS was used as positive control.

Samples were measured in duplicate, accompanied by duplicate calibratorwells containing a thrombin calibrator (containing a pre-determinedquantity of thrombin) in the same plasma. The 96 well plate was warmedto 37° C. in a Fluoroskan Ascent plate reader (Thermo) for 10 minutes.Thrombin generation commenced upon addition of 20 μl FluCa reagent(fluorogenic substrate, ZGGR-AMC (2.5 mM), in buffer containing 100 mMCaCl₂). TGA reagents were obtained from Stago. Increase in fluorescenceover time was monitored by the plate reader.

A thrombin calibrator curve was run alongside each sample beinginvestigated. Using a calibrator, with a known concentration ofthrombin, the amount of thrombin generated in a sample underinvestigation can be calculated from the fluorescent signal obtainedusing software ThrombinoscopeBV. Fluorescence from test wells wascalibrated against fluorescence from the thrombin calibrator wells, todetermine the equivalent thrombin generated in the test wells.

Run data were analysed using Stago analysis software. The amount ofthrombin generated was determined using the thrombin calibrator curvewith known activity. The following aspects of the thrombogram weredetermined: lag time (minutes), endogenous thrombin potential (ETP; areaunder the thrombogram, nM thrombin/minute), peak height (Cmax; nMthrombin), time to peak (Tmax/minutes), velocity index (VI; nM/minute,slope between lag time and time to peak) and tail start (minutes; timeat which the thrombin generation has come to an end).

Example 14. Dose Response and Potency in Thrombin Generation Assay

To evaluate the maximal thrombin peak height (Cmax, nM Thrombin) andtime to peak (Tmax, minutes) of bispecific antibody IXAX-1280.0999.0325we performed thrombin generation assays (TGA) in human FVIII-depletedplasma using a full antibody concentration dose response according tothe method set out in Example 13. Data generated from dose responsecurves was fitted using a non-linear log [antibody] vs responseparameter variable slope model (4 parameter logistic regression model).AbE was included for comparison. IXAX-1280.0999.0325 and AbE used inthis assay were determined by mass spectrometry to be close to 100%heterodimer, with no homodimeric contaminants detected.

Over a prospective therapeutic window spanning 300 to 30 nM, equivalentto 45 to 4.5 μg/ml, we observed equivalent (within 10%) or greater Cmax(nM Thrombin) values for IXAX-1280.0999.0325 compared to AbE at allconcentrations analysed (FIG. 30). Cmax of IXAX-1280.0999.0325 was closeto that of a normal plasma control when the concentrations of thebispecific antibody were between 100 and 300 nM (FIG. 30 and FIG. 31).In contrast, Cmax of AbE only reached the normal level when IgGconcentration is 300 nM. In this study, the EC50 of IXAX-1280.0999.0325(8.0 nM) was approximately 15% of the EC50 of AbE (54.4 nM).

Using the Cmax curve, it can be predicted that IXAX-1280.0999.0325 canachieve the same activity as 45 μg/mL of emicizumab when itsconcentration is equal to or greater than 8 μg/mL, which suggests apotential efficacy advantage with IXAX-1280.0999.0325 compared withemicizumab.

Analysis of the same dose response but with respect to Tmax, we observedequivalent (within 10%) or less than (or reduced) Tmax values forIXAX-1280.0999.0325 compared to AbE at all concentrations analysed (FIG.32). Calculated EC50 values based on Tmax values obtained are 1.65 nMfor IXAX-1280.0999.0325 (circle) compared to 2.8 nM for AbE (square).

In respect to the therapeutic ranges indicated in FIGS. 30 and 32, weobserve with IXAX-1280.0999.0325 Cmax and Tmax dose response curveswhich are greater and lower compared to AbE, respectively.

TABLE E14-1 Non-linear fit of Cmax. Best fit values for log of antibodyconcentration vs Cmax. Variable slope (4 parameters).IXAX-1280.0999.0325 Ab_E Bottom 18.20 4.053 Top 392.0 399.9 LogEC50−8.098 −7.264 HillSlope 1.111 1.135 EC50 7.984e−009 5.440e−008 Span373.8 395.8

TABLE E14-2 Non-linear fit of Tmax. Best fit values for log of antibodyconcentration vs Cmax. Variable slope (4 parameters).IXAX-1280.0999.0325 Ab_E Bottom 2.888 −0.2454 Top 37.14 62.16 LogEC50−8.781 −8.548 HillSlope −0.7713 −0.5577 EC50 1.654e−009 2.829e−009 Span34.25 62.41

The activities of three further bispecific antibodies (BiAb 2, 3 and 4)were also assessed in the TGA and compared against the performance ofIXAX-1280.0999.0325 (BiAb 1) and commercially available emicizumabcalibrator (Enzyme Research Laboratories) in commercially availablehuman FVIII-depleted plasma (Helena Biosciences). BiAbs were as follows,each including heavy chain constant regions SEQ ID NO: 409 and SEQ IDNO: 410 respectively in the two heavy chains, and lambda light chainconstant region SEQ ID NO: 146 in the common light chain:

-   1. IXAX-1280.0999.0325. Anti-FIX heavy chain SEQ ID NO: 419, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   2. IXAX-1454.0999.0325. Anti-FIX heavy chain SEQ ID NO: 424, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   3. IXAX-1441.0999.0325. Anti-FIX heavy chain SEQ ID NO: 426, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   4. IXAX-1442.0736.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX    heavy chain SEQ ID NO: 430, common light chain SEQ ID NO: 414.

BiAb_1, 2, 3 and 4 dose-dependently increased thrombin peak height(Cmax), and dose-dependently decreased time to peak (Tmax) in the samemanner as emicizumab. The top of Cmax curve of BiAb_1 was measured atabout 368 nM, higher than that of emicizumab (334.8 nM). EC50 (Cmax) ofBiAb_1, 2, 3, and 4 were similar to each other and had calculated EC50sof 6.45 nM, 5.87 nM, 5.2 nM and 4.81 nM respectively, representing EC50sbetween 26% and 35% of the EC50 of emicizumab (18.33 nM). FIG. 33 A.Calculated EC50 (Tmax) values for BiAb_1 to BiAb_4 were 0.56 nM, 0.65nM, 1.08 nM and 0.93 nM respectively, compared with 2.53 nM foremicizumab. FIG. 33 B.

In a third study, BiAb_1 (IXAX-1280.0999.0325) was again compared withcommercially available emicizumab calibrator by using TGA assay in humanFVIII-depleted plasma. BiAb_1 dose-dependently increased thrombin peakheight (Cmax), and dose-dependently decreased time to peak (Tmax). FIG.34. The top of Cmax curve for BiAb_1 was about 396.5 nM, higher thanthat of emicizumab (286.3 nM). EC50 of BiAb_1 was 7.7 nM, approximately30% of the EC50 of emicizumab (25.9 nM).

Assay to assay variation is observed between the TGA as shown in FIG.34, FIG. 33 and FIG. 30, in which BiAb_1 exhibited Cmax of approximately400 nM, 375 nM and 375 nM respectively and in which emicizumab exhibitedCmax of 275 nM, 300 nM and 350 nM. Despite variation in the absolutereadings, the trends observed were the same in each instance of the TGA.

In summary, TGA data with either the commercially available emicizumabcalibrator or the generated reference antibody AbE consistentlyindicated an efficacy advantage for BiAb_1 (IXAX-1280.0999.0325)compared with emicizumab. An advantage was also observed with the otherantibodies tested (BiAb_2, BiAb_3 and BiAb_4).

According to an FDA multi-disciplinary review of emicizumab, a medianannualized bleeding rate (ABR) of 0 would be achieved at emicizumabsteady state trough plasma concentration ≥45 μg/mL[17]. Using the Cmaxcurves from the TGA described above, it is predicted that BiAb_1 canachieve the same activity as 45 μg/mL of emicizumab when itsconcentration is equal to or greater than about 2-4 μg/mL. Thisobservation suggests a potential efficacy and/or dosing advantage withrespect to emicizumab. The differences in activity potentially mean thatthe bispecific antibody can achieve the same therapeutic effect whenadministered at lower dose and/or less frequently than emicizumab,representing a clinical advantage. Although the higher Cmax indicatesthe potential for a more powerful procoagulant capability, the magnitudeof this increase is unlikely to be associated with safety concerns.

Example 15. Affinities of Optimised Antibody Arms

Affinity and kinetics of purified anti-FIX and anti-FX antibodies forbinding to their respective antigens was determined by SPR as describedin Example 10 above.

The anti-FIX antibodies showed binding to FIX with an affinity range ofapproximately

0.05 μM-0.3 μM (50-300 nM), with a general trend of increasing affinity(lower KD) and faster off-rate correlating with greater activity in thebispecific antibody. Table E15-1.

TABLE E15-1 Captured anti-FIX IgG k_(on) (1/MS) k_(off) (1/s) K_(D) (M)Isotype control antibody No binding No binding No binding AbN 3.64 × 10⁴6.48 × 10⁻² 1.78 × 10⁻⁶ NINA-0128 1.37 × 10⁵ 4.92 × 10⁻² 3.59 × 10⁻⁷NINA-0436.0128 1.46 × 10⁵ 5.23 × 10⁻² 3.59 × 10⁻⁷ NINA-0511.0128 1.34 ×10⁵ 1.75 × 10⁻² 1.31 × 10⁻⁷ NINA-1091.0128 1.61 × 10⁵ 3.52 × 10⁻² 2.18 ×10⁻⁷ NINA-1172.0128 2.25 × 10⁵ 1.71 × 10⁻² 7.64 × 10⁻⁸ NINA-1280.01281.92 × 10⁵ 1.00 × 10⁻² 5.23 × 10⁻⁸

The anti-FX antibodies showed binding to FX with an affinity range ofapproximately 0.3-3 μM. Table E15-2. Anti-FX antibody MONA was includedas a control low affinity antibody with VH and VL domains from an IgMclone obtained from the single cell sorting (Example 1).

TABLE E15-2 Captured anti-FX IgG k_(on) (1/MS) k_(off) (1/s) K_(D) (M)Isotype control antibody No binding No binding No binding AbT 2.50 × 10⁴3.85 × 10⁻² 1.54 × 10⁻⁶ TINA-0200.0128 4.43 × 10⁴ 1.12 × 10⁻¹ 2.52 ×10⁻⁶ TINA-0201.0128 5.90 × 10⁴ 4.82 × 10⁻² 8.16 × 10⁻⁷ TINA-0202.01285.13 × 10⁴ 7.09 × 10⁻² 1.38 × 10⁻⁶ TINA-0616.0128 4.19 × 10⁴ 2.22 × 10⁻²5.30 × 10⁻⁷ TINA-0638.0128 6.99 × 10⁴ 2.32 × 10⁻² 3.33 × 10⁻⁷TINA-0666.0128 4.49 × 10⁴ 4.17 × 10⁻² 9.27 × 10⁻⁷ MONA_IgG4PE 5.36 × 10⁴5.24 × 10⁻² 9.78 × 10⁻⁷

Example 16. Initial Biophysical Assessment

To evaluate expression of the bispecific antibodies, IXAX-1172.0201.0128was chosen as a representative antibody for minipool analysis. Minipoolanalysis allows screening of CHO stably transfected cells expressinglarge amounts (at least 1 g/I) of heterodimeric bispecific antibody andrepresents a means of evaluating stable bispecific antibody expression.

Using standard Lonza fed-batch overgrowth protocols for stablytransfected CHO-K1 cells, bispecific antibodies were expressed. Aftertransfection, 5000 viable cells were aliquoted per well to generatemultiple minipools. 8 were taken forward based on antibody titres asmeasured by Octet.

Cells were harvested, filtered and purified by Protein A chromatographyto isolate the antibodies from the supernatant. Antibody concentration(mg) was quantified by OD280, total amount of antibody (mg) wascalculated accordingly based on volume of sample and a purificationyield (mg/L) assigned according to cell culture volume. The relativepercentages of heterodimer and homodimers in each of the 8 minipoolsamples was determined using imaged capillary isoelectic focusing(icIEF) (Protein Simple, Maurice). Homodimer and heterodimer peaks wereassigned using transiently expressed reference homodimer arms for FIXand FX.

We were able to isolate stably transfected cells expressingapproximately 1 g/L bispecific antibody with up to approximately 95%heterodimer (e.g., as shown for MP_1 and MP_7, FIG. 35).

Bispecific antibody activity in FXase assay correlated with %heterodimer with a Pearson's correlation coefficient of 0.99 (FIG. 36).

Example 17. Purification of Bispecific Antibody

Co-expression of the two heavy chains and one common light chain of abispecific antibody generates a composition comprising the bispecificantibody plus monospecific antibody byproducts. These may be separatedby ion exchange chromatography, exploiting differences in theisoelectric point of the bispecific heterodimer compared with themonospecific homodimers.

Bispecific antibody IXAX-1280.0999.0128 comprises anti-FIX heavy chainSEQ ID NO: 419, anti-FX heavy chain SEQ ID NO: 421 and common lightchain SEQ ID NO: 405. The bispecific antibody was purified followingco-expression of these polypeptides in HEK cells, using protein Achromotography to isolate the antibodies from cell supernatant, followedby ion exchange chromatography to isolate the heterodimer.

Bispecific antibody IXAX-0436.0201.0128 comprises anti-FIX heavy chaincomprising VH domain SEQ ID NO: 324 and an IgG4 human heavy chainconstant region with P (hinge) mutation and K439E, anti-FX heavy chaincomprising VH domain SEQ ID NO: 470 and IgG4 human heavy chain constantregion with P (hinge) mutation and E356K, and common light chain SEQ IDNO: 405.

Bispecific antibody IXAX-0436.0202.0128 comprises anti-FIX heavy chaincomprising VH domain SEQ ID NO: 324 and an IgG4 human heavy chainconstant region with P (hinge) mutation and K439E, anti-FX heavy chaincomprising VH domain SEQ ID NO: 472 and IgG4 human heavy chain constantregion with P (hinge) mutation and E356K, and common light chain SEQ IDNO: 405.

Bispecific antibody IXAX-1172.0201.0128 comprises anti-FIX heavy chaincomprising VH domain SEQ ID NO: 440 and an IgG4 human heavy chainconstant region with P (hinge) mutation and K439E, anti-FX heavy chaincomprising VH domain SEQ ID NO: 470 and an IgG4 human heavy chainconstant region with P (hinge) mutation and E356K, and common lightchain SEQ ID NO: 405.

Ion exchange chromatography cleanly separated each antibody compositioninto its component parts. Baseline separation was observed. Anti-FIXxFXheterodimeric bispecific antibody is separated from homodimericcontaminant anti-FIX and/or anti-FX monospecific antibodies.

FIG. 37a shows successful purification of IXAX-1280.0999.0128 from acomposition comprising the bispecific antibody mixed with anti-FIXhomodimer NINA-1280.0128 and anti-FX homodimer TINA-0999.0128.

FIG. 37b shows successful purification of IXAX-0436.0202.0128 from acomposition comprising the bispecific antibody mixed with anti-FIXhomodimer NINA-0436.0128 and anti-FX homodimer TINA-0202.0128. Thechromatogram represents cation ion exchange purification of N436bispecific antibody from homodimer contaminants using a series ofstepwise elutions using increasing concentrations of NaCl up to 500 nMin Sodium acetate pH 5. Peak 1 represents anti-FIX homodimer antibody;peak 2, anti-FIX/FX bispecific antibody and peak 3 represents anti-FXhomodimer antibody. Peak 1, 2 and 3 make up 18%, 79% and 3% total peakarea respectively.

FIG. 37c shows successful purification of IXAX-1172.0201.0128 from acomposition comprising the bispecific antibody and anti-FIX homodimerNINA-1172.0128. The column purification here yielded 31.5% anti-FIXhomodimer and 68.5% bispecific heterodimer. The chromatogram representscation ion exchange purification of N1172 bispecific antibody fromanti-FIX homodimer contaminants using an initial stepwise elution toremove weakly bound Peak 1 (anti-FIX homodimer) followed by a gradientelution using increasing concentrations of NaCl to elute the anti-FIX/FXbispecific. The presence of anti-FX homodimer was not detected.

Materials & Methods

For IXAX-1280.0999.0128 purification, bispecific antibody wastransiently expressed in Expi293F HEK cells. Cell culture supernatantwas harvested, filtered and loaded on to a 5 ml HiTrap MabSelect Sure(MSS) column (GE Healthcare) equilibrated with 1× phosphate bufferedsaline (PBS). The column was washed with 5 column volumes of PBS andbound antibody was eluted using IgG elute (ThermoFisher). Elutedbispecific antibody was dialysed into 1×PBS overnight at 4° C. andconcentrated using a centrifugal filter unit with a 10 kDa molecularweight cut off.

Chromatography was performed at room temperature. A 1 ml HiTrap Capto SPcolumn (GE Healthcare) was equilibrated with 20 mM sodium phosphate, pH6.0 and 0.5 mg of Protein A purified material, diluted 1:20 inequilibration buffer (20 mM sodium phosphate, pH 6.0), was loaded on tothe column. The column was subsequently washed with 10 column volumes ofequilibration buffer followed by a linear gradient (100% B over 90column volumes to 500 mM NaCl) to elute the bispecific antibody andmonospecific contaminants. In this process, buffer is progressivelychanged from A (20 mM sodium phosphate, pH 6.0, no salt) to B (buffer Awith the addition of 500 mM NaCl) over 90 cv at a flow rate of 1 ml/minfor the 1 ml column.

For IXAX-0436.0202.0128 purification, a stepwise gradient includingwashes at three different ionic strengths was applied using variedproportions of Buffer A (50 mM sodium acetate, pH 5) and Buffer B (50 nMsodium acetate and 500 mM sodium chloride).

For IXAX-1172.0201.0128 purification. an initial stepwise elution wasused to remove weakly bound Peak 1 (anti-FIX homodimer) followed by agradient elution using increasing concentrations of NaCl to elute theanti-FIX/FX bispecific.

Subsequently, the following bispecific antibodies were expressed in CHOcells:

-   5. IXAX-1280.0999.0325. Anti-FIX heavy chain SEQ ID NO: 419, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   6. IXAX-1454.0999.0325. Anti-FIX heavy chain SEQ ID NO: 424, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   7. IXAX-1441.0999.0325. Anti-FIX heavy chain SEQ ID NO: 426, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.-   8. IXAX-1442.0736.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX    heavy chain SEQ ID NO: 430, common light chain SEQ ID NO: 414.-   9. IXAX-1442.0687.0325. Anti-FIX heavy chain SEQ ID NO: 428, anti-FX    heavy chain SEQ ID NO: 421, common light chain SEQ ID NO: 414.

Each of these bispecific antibodies includes heavy chain constantregions SEQ ID NO:

409 and SEQ ID NO: 410 respectively for the two heavy chains, and lambdalight chain constant region SEQ ID NO: 146 in the common light chain.

The titres observed from transient expression of each of antibodies 1 to5 above in CHO cells were comparable to titres for a monospecificisotype control antibody. Stable pools and mini-pools (up to 4,000 cellsseeded after transfection) were also generated. Although the stablepools produced low percentages (11-19%) of heterodimeric antibody,mini-pools with titres up to 4.9 g/I and percentages of heterodimers upto 82% were established from a limited number of screened mini-pools.

After protein A purification, cation exchange chromatography was used toremove homodimeric by-products to generate high-purity materialssuitable for use in functional assays and for developability screening.

Using a gradient cation exchange method, antibodies 1 to 4 wereseparated from the homodimeric by-products, providing 91-96% heterodimerin the eluted material. Thus, even with this preliminary purificationmethod we were able to obtain 91-96% pure heterodimer. FIG. 38. Nocomparable homodimer/heterodimer separation was observed for antibody 5using this technique.

Example 18. Quality Assessment by Mass Spectrometry

The structural integrity of therapeutic monoclonal antibodies can becompromised by multiple types of post-translational modifications whichresult in product heterogeneity. Mass spectrometry (MS) was used tocharacterize and evaluate the quality of the bispecific antibodies aftercation exchange purification.

After cation exchange separation as described in Example 17, the threedifferent species (anti-FIX/anti-FX heterodimer, anti-FIX homodimer andanti-FX homodimer respectively) in the eluted composition of BiAb_1IXAX-1280.0999.0325 were analysed by MS. Molecular weights (MW) of thethree molecules determined by MS matched the theoretical MW predicted byamino acid sequences of BiAb_1. MS results thus confirmed the identityand the purity of FIX/FX heterodimer after cation exchange purification.

Example 19. Stability Assessment

After purification as described in Example 17, BiAb_1, 2, 3 and 4respectively were buffer exchanged to either buffer 1 (sodium acetate,pH 5.5) or buffer 2 (citrate/phosphate, pH 6.0), stored for 2 weeks at4° C., for 4 weeks at 25° C., or underwent 1× freeze/thaw cycle. Theconcentration of IgG was measured before and after treatment tocalculate the loss of antibodies due to the treatment. SEC-HPLC was alsoperformed before and after treatment to monitor for bispecific antibodydegradation and aggregation. No obvious loss or degradation of BiAb_1,2, 3 or 4 was observed. These four bispecific antibodies were thus allstable in both buffer 1 and buffer 2.

Example 20. Dose Response and Potency in FXase Assay

A FXase kinetic assay was conducted to measure the factor VIII mimeticactivity of IXAX-1280.0999.0325 and AbE in a dose response to determinetheir EC50 values as per Example 7. Data generated from dose responsecurves was fitted using a non-linear log[antibody] vs response parametervariable slope model (4 parameter logistic regression model). FIG. 39.The y-axis are plotted OD405 nm values at a 600 second assay timepoint.The EC50 values for both IXAX-1280.0999.0325 and AbE were calculatedfrom a non-linear regression curve of the data, giving an EC50 of 3.99nM for IXAX-1280.0999.0325 and EC50 of 261.2 nM for AbE. These areapproximations since the dose response curve was incomplete. Despitethis it is evident that IXAX-1280.0999.0325 has a significantly lowerEC50 than AbE. The activity of IXAX-1280.0999.0325 and AbE is dosedependent. IXAX-1280.0999.0325 demonstrated higher FVIII mimeticactivity, in particular at lower concentrations, compared with AbE.

TABLE E20-1 Kinetic FXase best fit values for log of antibodyconcentration vs response. Variable slope (4 parameters).IXAX-1280.0999.0325 Ab_E Bottom 0.1620 0.1103 Top 0.6872 0.9858 LogEC50−8.399 −6.583 HillSlope 2.707 1.087 EC50 3.993e−009 2.612e−007 Span0.5252 0.8756

Example 21. Hyphen Assay Dose Response

A chromogenic assay (HYPHEN BioMed), which analyses factor Xa productionin human plasma, was used to measure the factor VIII mimetic activity ofIXAX-1280.0999.0325 and AbE. In this assay, FXa generation isproportional to the OD405 measured after chromogenic substrate addition.Antibody concentration dose response curves were generated and fittedusing a non-linear log[antibody] vs response parameter variable slopemodel (4 parameter logistic regression model). EC50 values based on thedose responses were calculated. An EC50 value of 5.92 nM was calculatedfor IXAX-1280.0999.0325 compared with 15.43 nM for AbE.

IXAX-1280.0999.0325 consistently achieved greater FVIII mimetic abilitythan AbE over almost all concentrations. At 60 nM the A405 nm hadsaturated at 4.988 for both molecules. IXAX-1280.0999.0325 retained thissaturation at 20 nM while AbE presented with a decreased A405 nm of3.457. At the lowest concentration of 0.028 nM, IXAX-1280.0999.0325displayed over 4-fold greater absorbance than AbE. The calculated EC50values confirm that IXAX-1280.0999.0325 shows a superior potency whencomparing to AbE across a dose response assay. FIG. 40.

TABLE E21-1 Hyphen FXase EC50. Best fit values for log of antibodyconcentration vs response. Variable slope (4 parameters).IXAX-1280.0999.0325 Ab_E Bottom 0.7007 0.2581 Top 5.195 5.861 LogEC50−8.227 −7.812 HillSlope 1.848 1.231 EC50 5.924e−009 1.543e−008 Span4.495 5.603Materials & Methods

The BIOPHEN FVIII:C (Ref. 221402) kit was used following manufacture'sassay protocol. Briefly, FVIII deficient plasma (Helena BiosciencesEurope) was diluted 1:40 using Tris-BSA buffer (R4) and 45 μl was addedto a clear bottom 96-well plate. 5 μl of bispecific antibody was addedto the diluted plasma. 50 μl each of reagent R1 (FX) and R2 (FIXa),pre-incubated to 37° C., was added to each well and incubated at 37° C.for five minutes. Subsequently, 50 μl of reagent R3 (SXa-11, chromogenicreagent) was added, mixed and incubated for an additional five minutes,exactly. Addition of 50 μl 20% acetic acid terminated the reaction.Generation of Factor Xa was monitored through the ability of factor Xato cleave a specific factor Xa substrate (SXa-11). Cleavage of thissubstrate releases the coloured product, pNA, which can be monitoredusing a spectrophotometer at 405 nM and compared to a blank sample.

IXAX-1280.0999.0325 and AbE used in this assay were determined by massspectrometry to be close to 100% heterodimer, with no (or low levels of)homodimeric contaminants detected.

IXAX-1280.0999.0325 and AbE samples were diluted using a 1:3 dilutionseries with PBS as diluent. 5 μL volume was added to 45 μL factor VIIIdeficient plasma. The final concentrations (nM) of each sample in thedilution series (when assayed) were: 60.0, 20.0, 6.67, 2.22, 0.741,0.247, 0.082, and 0.028. Concentrations were converted to log(M) andplotted. A non-linear regression was plotted on the graph to enable EC50calculation.

Example 22. Dose Response and Potency in Plasma Coagulation Assay

We evaluated the activated partial thromboplastin time (aPTT) ofbispecific antibody AbE against IXAX-1280.0999.0325 using a fullantibody concentration dose response (method according to Example 8).Data generated from dose response curves were fitted using a non-linearlog[antibody] vs response parameter variable slope model (4 parameterlogistic regression model).

Over the concentration values analysed we observed equivalent aPTTvalues (within 10%) for IXAX-1280.0999.0325 compared to AbE at allconcentrations analysed (FIG. 41). Dose response curves were compared toa human IgG4 monoclonal antibody isotype control, which demonstrated nohaemostatic efficacy. Calculated EC50 values based on aPTT valuesobtained were 2.1 nM for IXAX-1280.0999.0325 (circle) compared with 2.0nM for AbE (square).

With respect to a prospective therapeutic range of 30-300 nM, we observewith IXAX-1280.0999.0325 an aPTT dose response curve equivalent to thatof AbE.

IXAX-1280.0999.0325 and AbE used in this assay were determined by massspectrometry to be 100% heterodimer, with no homodimeric contaminantsdetected.

Example 23. Activity in the Presence of Anti-FVIII Inhibitory Antibodies

Factor VIII replacement therapy can become ineffective for treatingpatients with haemophilia A if the patient develops alloantibodiesagainst the exogenously administered FVIII. Inhibitory anti-FVIIIalloantibodies may block the binding of FIX, phospholipid and vonWillebrand factor to FVIII, rendering it inactive.

Advantageously, therapeutic bispecific antibodies are insensitive to thepresence of FVIII alloantibodies in a patient's blood, as thealloantibodies have specificity to FVIII. This is confirmed by theability of a bispecific antibody to functionally restore haemostasis inplasma taken from an inhibitor patient. In this Example, we demonstratethis using two haemostatic assays: activated partial thromboplastin time(aPTT) and Thrombin Generation Assay (TGA) using plasma from a patientwith haemophilia A having inhibitory alloantibodies (referred to as“inhibitor plasma”). A restoration of clotting time indicated that thebispecific antibodies analysed are functional in the presence of a FVIIIinhibitory alloantibody. Thus, the data presented here indicate thatIXAX-1280.0999.0325 and IXAX-1441.0999.0325 will be able to functionallyrescue clotting time in patients who have inhibitory alloantibodiesagainst FVIII.

The Bethesda assay or the Nijmegen-Modified Bethesda assay is usedmeasure the titre of alloantibodies against FVIII. In these assays,different dilutions of patient's plasma are mixed with an equal volumeof ‘normal’ plasma and left to incubate for a period of time and thelevel of FVIII is measured. Presence of an inhibitor is indicated when adecrease in residual FVIII is observed. The unit of measurement in theseassays are known as Bethesda Units (BU)—a higher BU indicating greaterinhibition and lower residual FVIII activity. The experiments describedhere used patient plasma having a specific inhibitor level of 70 BU.

aPTT

IgG4 bispecific antibodies IXAX-1280.0999.0325, IXAX-1441.0999.0325 andAbE were expressed in CHO cells then purified by Protein Achromatography followed by cation exchange chromatography to separateactive heterodimer from contaminating homodimers and analysed at sixdifferent concentrations 300, 100, 33.3, 11.1, 3.7 and 1.23 nM by aPTT.aPTT was carried out as per Example 8. Data generated from dose responsecurves were fitted using a non-linear log[antibody] vs responseparameter variable slope model (4 parameter logistic regression model).Both IXAX-1280.0999.0325 and IXAX-1441.0999.0325 IgG4 antibodies wereable to rescue the clotting defect in the inhibitor patient sample in asimilar manner to AbE. FIG. 42.

Thrombin Generation Assay

Thrombin generation in inhibitor plasma (70 BU) was determined using forIXAX-1280.0999.0325, IXAX-1441.0999.0325 and AbE IgG4 bispecificantibodies following purification on Protein A followed by cationexchange chromatography. A thrombin generation assay was used as perExample 13. A thrombin peak was observed for all bispecific antibodies,indicating that both IXAX-1280.0999.0325 and IXAX-1441.0999.0325 canfunctionally restore haemostasis in plasma containing inhibitoryalloantibodies to FVIII, in a similar way to AbE.

A dose response for each bispecific antibody was carried out and thepeak thrombin height (Cmax) was determined. FIG. 43. In theconcentration range analysed, the Cmax dose responses forIXAX-1280.0999.0325 and IXAX-1441.0999.0325 were greater than the Cmaxof AbE, indicating greater thrombin burst, and the Tmax dose responsesfor IXAX-1280.0999.0325 and IXAX-1441.0999.0325 were lower than the Tmaxof AbE, indicating faster thrombin burst. FIG. 44.

REFERENCES

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FIXa Binding Arm VH Domain Polypeptide Sequences

TABLE S-9A Anti-FIXa VH domain sequences and CRDs VH amino Ab VH HCDR1HCDR2 HCDR3 VH nucleotide sequence acid sequence N0192H SEQ ID SEQ IDSEQ ID NO: 11 NO: 12 NO: 13GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFSSY IKQDGSE AREGYSSGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCT RLSCAASGFTFSSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAVRQAPGKGLEWVANIKQD VGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTC GSEKYYVDSVKGRFTISRCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCG DNAKNSLYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGCAGTTACTACTAC TAVYYCAREGYSSYYYYGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0212HSEQ ID SEQ ID SEQ ID SEQ ID NO: 16 SEQ ID NO: 17 NO: 11 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFSSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCT RLSCAASGFTFSSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTC GSEKYYVDSVKGRFTISRCAGAGACAACGCCAAGAACTCACTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSLYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0205HSEQ ID SEQ ID SEQ ID SEQ ID NO: 19 SEQ ID NO: 20 NO: 18 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFIFSSY INQDGSE AREGYSSGAGACTCTCCTGTGTAGCCTCTGGATTCATCTTTAGTAGCTATTGGATGAGCT RLSCVASGFIFSSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAATATAAATCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTC GSEKYYVDSVKGRFTISRCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCG DNAKNSLYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGCAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0211HSEQ ID SEQ ID SEQ ID SEQ ID NO: 21 SEQ ID NO: 22 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAACTCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0203HSEQ ID SEQ ID SEQ ID SEQ ID NO: 25 SEQ ID NO: 26 NO: 23 NO: 2 NO: 24GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNNY INQDGSE AREGYTDGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAACTATTGGATGAGCT RLSCAVSGFTFNNYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCATCATCTC GSEKFYVASVKGRFIISRCAGAGACAACGCCAAAAATTCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATACCGATTCGTCCTAT TAVYYCAREGYTDSSYYGTATGGAATGGACGTCTGGGGCCAAGGGACCACGGTCTCCGTCTCCTCA MDVWGQGTTVSVSS N0128HSEQ ID SEQ ID SEQ ID SEQ ID NO: 4 SEQ ID NO: 5 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG MDVWGQGTTVTVSS N0215HSEQ ID SEQ ID SEQ ID SEQ ID NO: 27 SEQ ID NO: 28 NO: 11 NO: 12 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFSSY IKQDGSE AREGYSSGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCT RLSCAASGFTFSSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAVRQAPGKGLEWVANIKQD VGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTC GSEKYYVDSVKGRFTISRCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCG DNAKNSLYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGCAGTTCGTCCTAC TAVYYCAREGYSSSSYYGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0216HSEQ ID SEQ ID SEQ ID SEQ ID NO: 29 SEQ ID NO: NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAACTCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0217HSEQ ID SEQ ID SEQ ID SEQ ID NO: 31 SEQ ID NO: NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAACTCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0218HSEQ ID SEQ ID SEQ ID SEQ ID NO: 33 SEQ ID NO: NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAAATCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKKSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0219HSEQ ID SEQ ID SEQ ID SEQ ID NO: 35 SEQ ID NO: 36 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAACTCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKNSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0220HSEQ ID SEQ ID SEQ ID SEQ ID NO: 37 SEQ ID NO: 38 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0221HSEQ ID SEQ ID SEQ ID SEQ ID NO: 39 SEQ ID NO: 40 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAACTCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKNSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0222HSEQ ID SEQ ID SEQ ID SEQ ID NO: 41 SEQ ID NO: 42 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKKSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0223HSEQ ID SEQ ID SEQ ID SEQ ID NO: 43 SEQ ID NO: 44 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0224HSEQ ID SEQ ID SEQ ID SEQ ID NO: 45 SEQ ID NO: 46 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAACTCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKNSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0225HSEQ ID SEQ ID SEQ ID SEQ ID NO: 47 SEQ ID NO: 48 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAAATCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKKSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0226HSEQ ID SEQ ID SEQ ID SEQ ID NO: 49 SEQ ID NO: 50 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAACTCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKNSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0227HSEQ ID SEQ ID SEQ ID SEQ ID NO: 51 SEQ ID NO: 52 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATCTACAAATGAACAGCCTGAGAGCCG DNAKKSVYLQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0228HSEQ ID SEQ ID SEQ ID SEQ ID NO: 53 SEQ ID NO: 54 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAACTCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKNSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N0229HSEQ ID SEQ ID SEQ ID SEQ ID NO: 55 SEQ ID NO: 56 NO: 1 NO: 2 NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT EVQLVESGGGLVQPGGSLGFTFNSY INQDGSE AREGYSSGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWW K SSYYGMD GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD VGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATCTC GSEKFYVASVKGRFTISRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTCCTAT TAVYYCAREGYSSSSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSSSEQ ID NO: SEQ ID NO: SEQ ID NO: 142 140 141 HCDR3 consensus HCDR1 HCDR2AREGYSSSSYYGMDV consensus consensus TDYY GFTFSSYWI NN INQDGSEKAREGY(S/T)(S/D)(S/Y)YYGMDV GF(T/I)F K (S/N)(S/N)YW I(N/K)QDGSEK N0420HSEQ ID SEQ ID SEQ ID SEQ ID NO: 238 SEQ ID NO: 314 NO: 1 NO: 2 NO: 161GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYAS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATGCCAGTTCGTCCTATTAVYYCAREGYASSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0421H SEQ ID SEQ ID SEQ ID SEQ ID NO: 239 SEQ ID NO: 315NO: 1 NO: 2 NO: 162GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSA GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTGCCTCGTCCTATTAVYYCAREGYSASSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0422H SEQ ID SEQ ID SEQ ID SEQ ID NO: 240 SEQ ID NO: 316NO: 1 NO: 2 NO: 163GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW ASYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTGCCTCCTATTAVYYCAREGYSSASYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0423H SEQ ID SEQ ID SEQ ID SEQ ID NO: 241 SEQ ID NO: 317NO: 1 NO: 2 NO: 164GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SAYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGGCCTATTAVYYCAREGYSSSAYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0430H SEQ ID SEQ ID SEQ ID SEQ ID NO: 242 SEQ ID NO: 318NO: 1 NO: 2 NO: 165GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW CSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTGCTCCTATTAVYYCAREGYSSCSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0431H SEQ ID SEQ ID SEQ ID SEQ ID NO: 243 SEQ ID NO: 319NO: 1 NO: 2 NO: 166GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW DSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTGACTCCTATTAVYYCAREGYSSDSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0432H SEQ ID SEQ ID SEQ ID SEQ ID NO: 244 SEQ ID NO: 320NO: 1 NO: 2 NO: 167GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW ESYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTGAGTCCTATTAVYYCAREGYSSESYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0433H SEQ ID SEQ ID SEQ ID SEQ ID NO: 245 SEQ ID NO: 321NO: 1 NO: 2 NO: 168GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW FSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTTCTCCTATTAVYYCAREGYSSFSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0434H SEQ ID SEQ ID SEQ ID SEQ ID NO: 246 SEQ ID NO: 322NO: 1 NO: 2 NO: 169GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW GSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTGGCTCCTATTAVYYCAREGYSSGSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0435H SEQ ID SEQ ID SEQ ID SEQ ID NO: 247 SEQ ID NO: 323NO: 1 NO: 2 NO: 170GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW HSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTCACTCCTATTAVYYCAREGYSSHSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0436H SEQ ID SEQ ID SEQ ID SEQ ID NO: 248 SEQ ID NO: 324NO: 1 NO: 2 NO: 171GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW ISYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCTCCTATTAVYYCAREGYSSISYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0437H SEQ ID SEQ ID SEQ ID SEQ ID NO: 249 SEQ ID NO: 325NO: 1 NO: 2 NO: 172GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW KSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTAAGTCCTATTAVYYCAREGYSSKSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0438H SEQ ID SEQ ID SEQ ID SEQ ID NO: 250 SEQ ID NO: 326NO: 1 NO: 2 NO: 173GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW LSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTCTGTCCTATTAVYYCAREGYSSLSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0439H SEQ ID SEQ ID SEQ ID SEQ ID NO: 251 SEQ ID NO: 327NO: 1 NO: 2 NO: 174GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW MSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATGTCCTATTAVYYCAREGYSSMSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0440H SEQ ID SEQ ID SEQ ID SEQ ID NO: 252 SEQ ID NO: 328NO: 1 NO: 2 NO: 175GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW NSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTAACTCCTATTAVYYCAREGYSSNSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0441H SEQ ID SEQ ID SEQ ID SEQ ID NO: 253 SEQ ID NO: 329NO: 1 NO: 2 NO: 176GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW PSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTCCCTCCTATTAVYYCAREGYSSPSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0442H SEQ ID SEQ ID SEQ ID SEQ ID NO: 254 SEQ ID NO: 340NO: 1 NO: 2 NO: 177GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW QSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTCAGTCCTATTAVYYCAREGYSSQSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0443H SEQ ID SEQ ID SEQ ID SEQ ID NO: 255 SEQ ID NO: 341NO: 1 NO: 2 NO: 178GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW RSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTAGATCCTATTAVYYCAREGYSSRSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0444H SEQ ID SEQ ID SEQ ID SEQ ID NO: 256 SEQ ID NO: 342NO: 1 NO: 2 NO: 179GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW TSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTACCTCCTATTAVYYCAREGYSSTSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0445H SEQ ID SEQ ID SEQ ID SEQ ID NO: 257 SEQ ID NO: 343NO: 1 NO: 2 NO: 180GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW VSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTGTGTCCTATTAVYYCAREGYSSVSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0446H SEQ ID SEQ ID SEQ ID SEQ ID NO: 258 SEQ ID NO: 344NO: 1 NO: 2 NO: 181GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW WSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTGGTCCTATTAVYYCAREGYSSWSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0447H SEQ ID SEQ ID SEQ ID SEQ ID NO: 259 SEQ ID NO: 345NO: 1 NO: 2 NO: 182GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW YSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTACTCCTATTAVYYCAREGYSSYSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0448H SEQ ID SEQ ID SEQ ID SEQ ID NO: 260 SEQ ID NO: 346NO: 1 NO: 2 NO: 183GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SCYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTGCTATTAVYYCAREGYSSSCYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0449H SEQ ID SEQ ID SEQ ID SEQ ID NO: 261 SEQ ID NO: 347NO: 1 NO: 2 NO: 184GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SDYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGGACTATTAVYYCAREGYSSSDYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0450H SEQ ID SEQ ID SEQ ID SEQ ID NO: 262 SEQ ID NO: 348NO: 1 NO: 2 NO: 185GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SEYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGGAGTATTAVYYCAREGYSSSEYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0451H SEQ ID SEQ ID SEQ ID SEQ ID NO: 263 SEQ ID NO: 349NO: 1 NO: 2 NO: 186GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SFYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTTCTATTAVYYCAREGYSSSFYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0452H SEQ ID SEQ ID SEQ ID SEQ ID NO: 264 SEQ ID NO: 350NO: 1 NO: 2 NO: 187GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SGYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGGGCTATTAVYYCAREGYSSSGYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0453H SEQ ID SEQ ID SEQ ID SEQ ID NO: 265 SEQ ID NO: 351NO: 1 NO: 2 NO: 188GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SHYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGCACTATTAVYYCAREGYSSSHYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0454H SEQ ID SEQ ID SEQ ID SEQ ID NO: 266 SEQ ID NO: 352NO: 1 NO: 2 NO: 189GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SIYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGATCTATTAVYYCAREGYSSSIYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0455H SEQ ID SEQ ID SEQ ID SEQ ID NO: 267 SEQ ID NO: 353NO: 1 NO: 2 NO: 190GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SKYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGAAGTATTAVYYCAREGYSSSKYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0456H SEQ ID SEQ ID SEQ ID SEQ ID NO: 268 SEQ ID NO: 354NO: 1 NO: 2 NO: 191GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SLYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGCTGTATTAVYYCAREGYSSSLYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0457H SEQ ID SEQ ID SEQ ID SEQ ID NO: 269 SEQ ID NO: 355NO: 1 NO: 2 NO: 192GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SMYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGATGTATTAVYYCAREGYSSSMYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0458H SEQ ID SEQ ID SEQ ID SEQ ID NO: 270 SEQ ID NO: 356NO: 1 NO: 2 NO: 193GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SNYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGAACTATTAVYYCAREGYSSSNYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0459H SEQ ID SEQ ID SEQ ID SEQ ID NO: 271 SEQ ID NO: 357NO: 1 NO: 2 NO: 194GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SPYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGCCCTATTAVYYCAREGYSSSPYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0460H SEQ ID SEQ ID SEQ ID SEQ ID NO: 272 SEQ ID NO: 358NO: 1 NO: 2 NO: 195GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SQYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGCAGTATTAVYYCAREGYSSSQYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0461H SEQ ID SEQ ID SEQ ID SEQ ID NO: 273 SEQ ID NO: 359NO: 1 NO: 2 NO: 196GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SRYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGAGATATTAVYYCAREGYSSSRYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0462H SEQ ID SEQ ID SEQ ID SEQ ID NO: 274 SEQ ID NO: 360NO: 1 NO: 2 NO: 197GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW STYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGACCTATTAVYYCAREGYSSSTYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0463H SEQ ID SEQ ID SEQ ID SEQ ID NO: 275 SEQ ID NO: 361NO: 1 NO: 2 NO: 198GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SVYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGGTGTATTAVYYCAREGYSSSVYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0464H SEQ ID SEQ ID SEQ ID SEQ ID NO: 276 SEQ ID NO: 362NO: 1 NO: 2 NO: 199GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SWYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTGGTATTAVYYCAREGYSSSWYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0465H SEQ ID SEQ ID SEQ ID SEQ ID NO: 277 SEQ ID NO: 363NO: 1 NO: 2 NO: 200GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SYYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTTCGTACTATTAVYYCAREGYSSSYYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0467H SEQ ID SEQ ID SEQ ID SEQ ID NO: 278 SEQ ID NO: 364NO: 1 NO: 2 NO: 201GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYCS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATTGCAGTTCGTCCTATTAVYYCAREGYCSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0468H SEQ ID SEQ ID SEQ ID SEQ ID NO: 279 SEQ ID NO: 365NO: 1 NO: 2 NO: 202GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYDS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATGACAGTTCGTCCTATTAVYYCAREGYDSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0469H SEQ ID SEQ ID SEQ ID SEQ ID NO: 280 SEQ ID NO: 366NO: 1 NO: 2 NO: 203GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYES GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATGAGAGTTCGTCCTATTAVYYCAREGYESSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0470H SEQ ID SEQ ID SEQ ID SEQ ID NO: 281 SEQ ID NO: 367NO: 1 NO: 2 NO: 204GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYFS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATTTCAGTTCGTCCTATTAVYYCAREGYFSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0471H SEQ ID SEQ ID SEQ ID SEQ ID NO: 282 SEQ ID NO: 368NO: 1 NO: 2 NO: 205GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYGS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATGGCAGTTCGTCCTATTAVYYCAREGYGSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0472H SEQ ID SEQ ID SEQ ID SEQ ID NO: 283 SEQ ID NO: 369NO: 1 NO: 2 NO: 206GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYHS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATCACAGTTCGTCCTATTAVYYCAREGYHSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0473H SEQ ID SEQ ID SEQ ID SEQ ID NO: 284 SEQ ID NO: 370NO: 1 NO: 2 NO: 207GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYIS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATATCAGTTCGTCCTATTAVYYCAREGYISSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0474H SEQ ID SEQ ID SEQ ID SEQ ID NO: 285 SEQ ID NO: 371NO: 1 NO: 2 NO: 208GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYKS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAAGAGTTCGTCCTATTAVYYCAREGYKSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0465H SEQ ID SEQ ID SEQ ID SEQ ID NO: 286 SEQ ID NO: 372NO: 1 NO: 2 NO: 209GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYLS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATCTGAGTTCGTCCTATTAVYYCAREGYLSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0476H SEQ ID SEQ ID SEQ ID SEQ ID NO: 287 SEQ ID NO: 373NO: 1 NO: 2 NO: 210GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYMS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATATGAGTTCGTCCTATTAVYYCAREGYMSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0477H SEQ ID SEQ ID SEQ ID SEQ ID NO: 288 SEQ ID NO: 374NO: 1 NO: 2 NO: 211GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYNS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAACAGTTCGTCCTATTAVYYCAREGYNSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0478H SEQ ID SEQ ID SEQ ID SEQ ID NO: 289 SEQ ID NO: 375NO: 1 NO: 2 NO: 212GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYPS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATCCCAGTTCGTCCTATTAVYYCAREGYPSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0479H SEQ ID SEQ ID SEQ ID SEQ ID NO: 290 SEQ ID NO: 376NO: 1 NO: 2 NO: 213GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYQS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATCAGAGTTCGTCCTATTAVYYCAREGYQSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0480H SEQ ID SEQ ID SEQ ID SEQ ID NO: 291 SEQ ID NO: 377NO: 1 NO: 2 NO: 214GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYRS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGAAGTTCGTCCTATTAVYYCAREGYRSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0481H SEQ ID SEQ ID SEQ ID SEQ ID NO: 292 SEQ ID NO: 378NO: 1 NO: 2 NO: 215GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYTS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATACCAGTTCGTCCTATTAVYYCAREGYTSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0482H SEQ ID SEQ ID SEQ ID SEQ ID NO: 293 SEQ ID NO: 379NO: 1 NO: 2 NO: 216GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYVS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATGTGAGTTCGTCCTATTAVYYCAREGYVSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0483H SEQ ID SEQ ID SEQ ID SEQ ID NO: 294 SEQ ID NO: 380NO: 1 NO: 2 NO: 217GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYWS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATTGGAGTTCGTCCTATTAVYYCAREGYWSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0484H SEQ ID SEQ ID SEQ ID SEQ ID NO: 295 SEQ ID NO: 381NO: 1 NO: 2 NO: 218GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYYS GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATTACAGTTCGTCCTATTAVYYCAREGYYSSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0485H SEQ ID SEQ ID SEQ ID SEQ ID NO: 296 SEQ ID NO: 382NO: 1 NO: 2 NO: 219GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSC GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTTGCTCGTCCTATTAVYYCAREGYSCSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0486H SEQ ID SEQ ID SEQ ID SEQ ID NO: 297 SEQ ID NO: 383NO: 1 NO: 2 NO: 220GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSD GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTGACTCGTCCTATTAVYYCAREGYSDSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0487H SEQ ID SEQ ID SEQ ID SEQ ID NO: 298 SEQ ID NO: 384NO: 1 NO: 2 NO: 221GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSE GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTGAGTCGTCCTATTAVYYCAREGYSESSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0488H SEQ ID SEQ ID SEQ ID SEQ ID NO: 299 SEQ ID NO: 385NO: 1 NO: 2 NO: 223GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSF GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTTTCTCGTCCTATTAVYYCAREGYSFSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0489H SEQ ID SEQ ID SEQ ID SEQ ID NO: 300 SEQ ID NO: 386NO: 1 NO: 2 NO: 224GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSG GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTGGCTCGTCCTATTAVYYCAREGYSGSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0490H SEQ ID SEQ ID SEQ ID SEQ ID NO: 301 SEQ ID NO: 387NO: 1 NO: 2 NO: 225GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSH GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTCACTCGTCCTATTAVYYCAREGYSHSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0491H SEQ ID SEQ ID SEQ ID SEQ ID NO: 302 SEQ ID NO: 388NO: 1 NO: 2 NO: 226GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSI GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTATCTCGTCCTATTAVYYCAREGYSISSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0492H SEQ ID SEQ ID SEQ ID SEQ ID NO: 303 SEQ ID NO: 389NO: 1 NO: 2 NO: 227GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSK GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAAGTCGTCCTATTAVYYCAREGYSKSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0493H SEQ ID SEQ ID SEQ ID SEQ ID NO: 304 SEQ ID NO: 390NO: 1 NO: 2 NO: 228GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSL GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTCTGTCGTCCTATTAVYYCAREGYSLSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0494H SEQ ID SEQ ID SEQ ID SEQ ID NO: 305 SEQ ID NO: 391NO: 1 NO: 2 NO: 229GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSM GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTATGTCGTCCTATTAVYYCAREGYSMSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0495H SEQ ID SEQ ID SEQ ID SEQ ID NO: 306 SEQ ID NO: 392NO: 1 NO: 2 NO: 230GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSN GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAACTCGTCCTATTAVYYCAREGYSNSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0496H SEQ ID SEQ ID SEQ ID SEQ ID NO: 307 SEQ ID NO: 393NO: 1 NO: 2 NO: 231GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSP GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTCCCTCGTCCTATTAVYYCAREGYSPSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0497G SEQ ID SEQ ID SEQ ID SEQ ID NO: 308 SEQ ID NO: 394NO: 1 NO: 2 NO: 232GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSQ GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTCAGTCGTCCTATTAVYYCAREGYSQSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0498H SEQ ID SEQ ID SEQ ID SEQ ID NO: 309 SEQ ID NO: 395NO: 1 NO: 2 NO: 233GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSR GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGATCGTCCTATTAVYYCAREGYSRSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0499H SEQ ID SEQ ID SEQ ID SEQ ID NO: 310 SEQ ID NO: 396NO: 1 NO: 2 NO: 234GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYST GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTACCTCGTCCTATTAVYYCAREGYSTSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0500H SEQ ID SEQ ID SEQ ID SEQ ID NO: 311 SEQ ID NO: 397NO: 1 NO: 2 NO: 235GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSV GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTGTGTCGTCCTATTAVYYCAREGYSVSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0501H SEQ ID SEQ ID SEQ ID SEQ ID NO: 312 SEQ ID NO: 398NO: 1 NO: 2 NO: 236GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSW GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTTGGTCGTCCTATTAVYYCAREGYSWSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS N0502H SEQ ID SEQ ID SEQ ID SEQ ID NO: 313 SEQ ID NO: 399NO: 1 NO: 2 NO: 237GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLAREGYSY GAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCTRLSCAVSGFTFNSYWMSW SSYYGMDGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDV GATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSEKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTTACTCGTCCTATTAVYYCAREGYSYSSYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGMDVWGQGTTVTVSS SEQ ID NO: 400 Consensus HCDR3 of N436 and selectedvariants with parent N128H AREGYSSXSYYGMDVX is I, L, V, R, W, Q, K, H, E, N, M, SRepresenting the N436H CDR3 sequence AREGYSSISYYGMDV inwhich the Ile is retained or replaced by Leu, Val, Arg, Trp,Gln, Lys, His, Glu, Asn, Met or Ser.SEQ ID NO: 401 Consensus HCDR3 of N436 and selectedvariants AREGYSSXSYYGMDV X is I, L, V, R, W, Q, K, H, E, N or MRepresenting the N436H CDR3 sequence AREGYSSISYYGMDV inwhich the Ile is retained or replaced by Leu, Val, Arg, Trp,Gln, Lys, His, Glu, Asn or Met.SEQ ID NO: 402 Consensus HCDR3 of N436 and selectedhydrophobic or positively charged variants AREGYSSXSYYGMDVX is I, L, V, R, W, Q or KRepresenting the N436H CDR3 sequence AREGYSSISYYGMDV inwhich the Ile is retained or replaced by Leu, Val, Arg, Trp, Gln or Lys.SEQ ID NO: 403 Consensus HCDR3 of initial most activevariants AREGYSSXSYYGMDV X is I, L or VRepresenting the N436M CDR3 sequence AREGYSSISYYGMDV inwhich the Ile is retained or replaced by Leu or Val.SEQ ID NO: 406 Consensus HCDR1 GFXGNSYW X is T or RRepresenting the N1280H CDR1 sequence GFRFNSYW (SEQ ID NO: 441) in which the Arg isretained or replaced by Thr. SEQ ID NO: 407 Consensus HCDR2 INQX₁GX₂X₃KX1 is D, G or W. X2 is S or F. X3 is E or R.Representing the N1280H CDR2 sequence INDGSRK (SEQ ID NO: 436) in which the Asp isretained or replaced by Gly or Trp, the Ser is retained or replaced by Phe and theArg is retained or replaced by Glu. SEQ ID NO: 634 Consensus HCDR2INQDGSXK X is R or E.Representing the N1280H CDR2 sequence INQDGSRK (SEQ ID NO: 436) in which the Arg isretained or replaced by Glu. SEQ ID NO: 408 Consensus HCDR3AREGYSSX₁X₂YYGMDV X1 is S or I. X2 is S or K.Representing the N1280H CDR3 sequence AREGYSSIKYYGMDV (SEQ ID NO: 433) inwhich the Ile is retained or replaced by Ser and the Lys is retained orreplaced by Ser. SEQ ID NO: 635 Consensus HCDR3 AREGYSSIXYYGMDVX is K or S.Reprsenting the N1280H CDR3 sequence AREGYSSIKYYGMDV (SEQ ID NO: 433) inwhich the Lys is retained or replaced by Ser. N0511H SEQ ID SEQ IDSEQ ID SEQ ID NO: 434 SEQ ID NO: 435 NO: 1 NO: 2 NO: 433GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANIKQDGATGGAAGTGAGAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSEKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTAT TAVYYCAREGYSSIKYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N1091HSEQ ID SEQ ID SEQ ID SEQ ID NO: 437 SEQ ID NO: 438 NO: 1 NO: 436 NO: 171GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDGATGGAAGTAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSRKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCTCCTAT TAVYYCAREGYSISSYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N1172HSEQ ID SEQ ID SEQ ID SEQ ID NO: 439 SEQ ID NO: 440 NO: 1 NO: 436 NO: 433GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTTAATAGCTATTGGATGAGCT RLSCAVSGFTFNSYWMSWGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDGATGGAAGTAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSRKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTAT TAVYYCAREGYSSIKYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N1280HSEQ ID SEQ ID SEQ ID SEQ ID NO: 442 SEQ ID NO: 443 NO: 441 NO: 436NO: 433 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCTEVQLVESGGGFVQPGGSL GAGACTCTCCTGTGCAGTCTCTGGATTCAGATTTAATAGCTATTGGATGAGCTRLSCAVSGFRFNSYWMSW GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQD GATGGAAGTAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSRKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTATTAVYYCAREGYSSIKYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAMDVWGQGTTVTVSS N1314H SEQ ID SEQ ID SEQ ID SEQ ID NO: 445 SEQ ID NO: 446NO: 441 NO: 444 NO: 433GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLINQGGS GAGACTCTCCTGTGCAGTCTCTGGATTCAGATTTAATAGCTATTGGATGAGCTRLSCAVSGFRFNSYWMSW RKGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQGGGCGGAAGTAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GSRKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTAT TAVYYCAREGYSSIKYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N1327HSEQ ID SEQ ID SEQ ID SEQ ID NO: 448 SEQ ID NO: 449 NO: 441 NO: 447NO: 433 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCTEVQLVESGGGFVQPGGSL INQWGSGAGACTCTCCTGTGCAGTCTCTGGATTCAGATTTAATAGCTATTGGATGAGCT RLSCAVSGFRFNSYWMSWRK GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAAVRQAPGKGLEWVANINQW TGGGGAAGTAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTCGSRKFYVASVKGRFTMSR CAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCGDNAKKSVYVQMNSLRAED AGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTATTAVYYCAREGYSSIKYYG TATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAMDVWGQGTTVTVSS N1333H SEQ ID SEQ ID SEQ ID SEQ ID NO: 451 SEQ ID NO: 452NO: 441 NO: 450 NO: 433GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTTGTCCAGCCTGGGGGGTCCCT EVQLVESGGGFVQPGGSLINQDGF GAGACTCTCCTGTGCAGTCTCTGGATTCAGATTTAATAGCTATTGGATGAGCTRLSCAVSGFRFNSYWMSW RKGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCAA VRQAPGKGLEWVANINQDGATGGATTCAGAAAATTCTATGTGGCCTCTGTGAAGGGCCGATTCACCATGTC GFRKFYVASVKGRFTMSRCAGAGACAACGCCAAGAAATCAGTGTATGTACAAATGAACAGCCTGAGAGCCG DNAKKSVYVQMNSLRAEDAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGTATAGTAGTATCAAGTAT TAVYYCAREGYSSIKYYGTATGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA MDVWGQGTTVTVSS N1454HSEQ ID SEQ ID SEQ ID SEQ ID NO: 453 SEQ ID NO: 454 NO: 441 NO:436NO: 433 GAGGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTEVQLVESGGGFVQPGGSL GAGACTGAGCTGTGCCGTGTCCGGCTTCCGGTTCAACAGCTACTGGATGTCCTRLSCAVSGFRFNSYWMSW GGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGCCAACATCAACCAGVRQAPGKGLEWVANINQD GACGGCAGCCGGAAGTTTTACGTGGCCTCTGTGAAGGGCAGATTCACCATGAGGSRKFYVASVKGRFTMSR CCGGGACAACGCCAAGAAAGAGGTGTACGTGCAGATGAACAGCCTGAGAGCCGDNAKKEVYVQMNSLRAED AGGACACCGCCGTGTACTATTGTGCCAGAGAGGGCTACAGCAGCATCAAGTACTAVYYCAREGYSSIKYYG TACGGCATGGACGTGTGGGGCCAGGGCACAACAGTGACAGTCTCTTCTMDVWGQGTTVTVSS N1441H SEQ ID SEQ ID SEQ ID SEQ ID NO: 455 SEQ ID NO: 456NO: 441 NO: 436 NO: 433GAGGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCT EVQLVESGGGFVQPGGSLGAGACTGAGCTGTGCCGTGTCCGGCTTCCGGTTCAACAGCTACTGGATGTCCT RLSCAVSGFRFNSYWMSWGGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGCCAACATCAACCAG VRQAPGKGLEWVANINQDGACGGCAGCCGGAAGTTTTACGTGGCCTCTGTGAAGGGCAGATTCACCATGAG GSRKFYVASVKGRFTMSRCCGGGACAACGCCGACAAAAGCGTGTACGTGCAGATGAACAGCCTGAGAGCCG DNADKSVYVQMNSLRAEDAGGACACCGCCGTGTACTATTGTGCCAGAGAGGGCTACAGCAGCATCAAGTAC TAVYYCAREGYSSIKYYGTACGGCATGGACGTGTGGGGCCAGGGCACAACAGTGACAGTCTCTTCT MDVWGQGTTVTVSS N1442HSEQ ID SEQ ID SEQ ID SEQ ID NO: 457 SEQ ID NO: 458 NO: 441 NO: 436NO: 433 GAGGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTEVQLVESGGGFVQPGGSL GAGACTGAGCTGTGCCGTGTCCGGCTTCCGGTTCAACAGCTACTGGATGTCCTRLSCAVSGFRFNSYWMSW GGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGCCAACATCAACCAGVRQAPGKGLEWVANINQD GACGGCAGCCGGAAGTTTTACGTGGCCTCTGTGAAGGGCAGATTCACCATGAGGSRKFYVASVKGRFTMSR CCGGGACAACGCCGAGAAAAGCGTGTACGTGCAGATGAACAGCCTGAGAGCCGDNAEKSVYVQMNSLRAED AGGACACCGCCGTGTACTATTGTGCCAGAGAGGGCTACAGCAGCATCAAGTACTAVYYCAREGYSSIKYYG TACGGCATGGACGTGTGGGGCCAGGGCACAACAGTGACAGTCTCTTCTMDVWGQGTTVTVSS

TABLE S-9B Anti-FIXa VH domain framework sequences Ab VH FR1 FR2 FR3 FR4N0192H SEQ ID NO: 148 SEQ ID NO: 133 SEQ ID NO: 149 SEQ ID NO: 135EVQLVESGGGLVQP YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC GGSLRLSCAAS N0212HSEQ ID NO: 148 SEQ ID NO: 133 SEQ ID NO: 149 SEQ ID NO: 135 N0205HSEQ ID N: 150 SEQ ID NO: 133 SEQ ID NO: 149 SEQ ID NO: 135EVQLVESGGGLVQP GGSLRLSCVAS N0211H SEQ ID NO: 151 SEQ ID NO: 133SEQ ID NO: 152 SEQ ID NO: 135 EVQLVESGGGLVQPFYVASVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYC GGSLRLSCAVS N0203H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 153 SEQ ID NO: 154FYVASVKGRFIISRDNAKNSVYLQMNSLRAEDTAVYYC WGQGTTVSVSS N0128H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 EVQLVESGGGFVQPMSWVRQAPGKGLEWVAN FYVASVKGRFTMSRDNAKKSVYVQMNSLRAEDTAVYYC WGQGTTVTVSSGGSLRLSCAVS N0215H SEQ ID NO: 148 SEQ ID NO: 133 SEQ ID NO: 149SEQ ID NO: 135 N0216H SEQ ID NO: 132 SEQ ID NO: 133 SEQ ID NO: 152SEQ ID NO: 135 N0217H SEQ ID NO: 151 SEQ ID NO: 133 SEQ ID NO: 155SEQ ID NO: 135 FYVASVKGRFTMSRDNAKNSVYLQMNSLRAEDTAVYYC N0218HSEQ ID NO: 151 SEQ ID NO: 133 SEQ ID NO: 156 SEQ ID NO: 135FYVASVKGRFTISRDNAKKSVYLQMNSLRAEDTAVYYC N0219H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 157 SEQ ID NO: 135FYVASVKGRFTISRDNAKNSVYVQMNSLRAEDTAVYYC N0220H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 158 SEQ ID NO: 135FYVASVKGRFTISRDNAKKSVYVQMNSLRAEDTAVYYC N0221H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 159 SEQ ID NO: 135FYVASVKGRFTMSRDNAKNSVYVQMNSLRAEDTAVYYC N0222H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 160 SEQ ID NO: 135FYVASVKGRFTMSRDNAKKSVYLQMNSLRAEDTAVYYC N0223H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N0224H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 155 SEQ ID NO: 135 N0225H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 156 SEQ ID NO: 135 N0226H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 157 SEQ ID NO: 135 N0227H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 160 SEQ ID NO: 135 N0228H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 159 SEQ ID NO: 135 N0229H SEQ ID NO: 151SEQ ID NO: 133 SEQ ID NO: 158 SEQ ID NO: 135 N0511H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1091H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1172H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1280H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1314H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1327H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1333H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO: 135 N1441H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 459 SEQ ID NO: 135FYVASVKGRFTMSRDNADKSVYVQMNSLRAEDTAVYYC N1442H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 460 SEQ ID NO: 135FYVASVKGRFTMSRDNAEKSVYVQMNSLRAEDTAVYYC N1454H SEQ ID NO: 132SEQ ID NO: 133 SEQ ID NO: 461 SEQ ID NO: 136FYVASVKGRFTMSRDNAKKEVYVQMNSLRAEDTAVYYC

FX Binding Arm VH Domain Polypeptide Sequences

TABLE S-10A Anti-FX VH domain sequences and CDRs VH amino Ab VH HCDR1HCDR2 HCDR3 VH nucleotide sequence acid sequence T02 SEQ ID SEQ IDSEQ ID SEQ ID NO: 60 SEQ ID NO: 61 NO: 57 NO: 58 NO: 59CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGC QVQLVQSGAEVKRPGASVGYTFTNY INAGNGF ARDWAAA CTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAACTKVSCKASGYTFTNYAIHW A T ISYYGMDATGCTATACATTGGGTGCGCCAGGCCCCCGGACAGAGGCTTGAGTGG VRQAPGQRLEWMGWINAG VATGGGATGGATCAACGCTGGCAATGGTTTCACAAAATCTTCACAGAA NGFTKSSQKFRGRVTITRGTTCCGGGGCAGAGTCACCATTACCAGGGACACATCCGCGAACACAG DTSANTAYMELSSLRSEDCCTACATGGAACTGAGCAGCCTCAGATCTGAAGACACGGCTATTTAT TAIYYCARDWAAAISYYGTACTGTGCGAGAGATTGGGCTGCTGCTATCTCTTACTACGGTATGGA MDVWGQGTTVTVSSCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG T05 SEQ ID SEQ ID SEQ IDSEQ ID NO: 70 SEQ ID NO: 71 NO: 67 NO: 68 NO: 69CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG QVQLVESGGGVVQPGRSLGFTFSSY IWYDGTN ARSGYSS GTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTRLSCAASGFTFSSYGMHW G K SWYGAMDATGGCATGCACTGGGTCCGCCAGGCTCCAGGCGAGGGGCTGGAGTGG VRQAPGEGLEWVAVIWYD VGTGGCAGTTATATGGTATGATGGAACTAATAAATACTATGCAGACTC GTNKYYADSLKGRFTISRCTTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC DNSKNTLYLQMNRLRAEDTCTATCTGCAAATGAACAGGCTGAGAGCCGAGGACACGGCTGTGTAT TAVYYCARSGYSSSWYGATACTGTGCGAGGTCCGGGTATAGCAGCAGCTGGTACGGCGCTATGGA MDVWGQGTTVTVSSCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG T06 SEQ ID SEQ ID SEQ IDSEQ ID NO: 80 SEQ ID NO: 81 NO: 77 NO: 78 NO: 79CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGC QVQLVQSGAEVKRPGASVGYTFTSY INAGNGI ARDWAAA CTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACAAGCTKVSCKASGYTFTSYAIHW A T ITYYGMDACGCCATACATTGGGTGCGCCAGGCCCCCGGACAGAGGCTTGAGTGG VRQAPGQRLEWMGWINAG VATGGGATGGATCAACGCTGGCAATGGTATCACAAAATCTTCACAGAA NGITKSSQKFQGRVTITRGTTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAACACAG DTSANTVYLELSSLRSEDTTTACCTGGAACTGAGCAGCCTCAGATCTGAAGACACGGCTGTTTAT TAVYYCARDWAAAITYYGTATTGTGCGAGAGATTGGGCTGCTGCTATCACCTACTACGGTATGGA MDVWGQGTTVTVSSCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG T12 SEQ ID SEQ ID SEQ IDSEQ ID NO: 89 SEQ ID NO: 90 NO: 86 NO: 87 NO: 88CAGGTGCAGCTGGTGGAGTCTGGGGGGGGCGTACTCCAGCCTGGGAA QVQLVESGGGVLQPGKSLEFTFSTA ISYDGSN AKDFTMV GTCCCTGAGACTCTCCTGTGCAGCCTCTGAATTCACCTTCAGTACCGRLSCAASEFTFSTAGMHW G K RGVIIMDCTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGG VRQAPGKGLEWVTFISYD VGTGACTTTTATATCATATGATGGAAGTAATAAATACTATGCAGACTC GSNKYYADSVKGRFTISRCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGGTGTATC DNSKVYLQMNSLRTEDTATGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGT VYYCAKDFTMVRGVIIMDGCGAAAGATTTCACTATGGTTCGGGGAGTTATTATAATGGACGTCTG VWGQGTTVTVSSGGGCCAAGGGACCACGGTCACCGTCTCCTCAG T14 SEQ ID SEQ ID SEQ ID SEQ ID NO: 99SEQ ID NO: 100 NO: 96 NO: 97 NO: 98CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA QVQLQESGPGLVKPSETLGGSISSY IYYSGST AKGAAGD GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTSLTCTVSGGSISSYYWSW Y Y ATTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGIRQPPGKGLEWIGYIYYS ATTGGGTATATCTATTACAGTGGGAGCACCAACTATAACCCCTCCCTGSTNYNPSLKSRVNISVD CAAGAGTCGAGTCAACATATCAGTAGACACGTCCAAGAACCAGTTCTTSKNQFSLRLSSVTAADT CCCTGAGGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTATAVYYCAKGAAGDYWGQGT TGTGCGAAAGGGGCAGCTGGGGACTACTGGGGCCAGGGAACCCTGGTLVTVSS CACCGTCTCCTCAG T15 SEQ ID SEQ ID SEQ ID SEQ ID NO: 108SEQ ID NO: 109 NO: 105 NO: 106 NO: 107CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA QVQLQESGPGLVKPSETLGGSISKY IYYSGNT ARGLGDY GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAAATSLTCTVSGGSISKYYWSW Y ACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGIRQPPGKGLEWIGYIYYS ATTGGATATATCTATTACAGTGGGAACACCTACCAGAATCCCTCCCTGNTYQNPSLKSRVTISID  CAAGAGTCGAGTCACCATATCAATAGACACGTCCAAGAACCAGATCTTSKNQISLKVSSVTAADT CCCTGAAGGTGAGCTCTGTGACCGCTGCGGACACGGCCGTCTATTACAVYYCARGLGDYWGQGTL TGTGCGAGAGGGCTGGGGGACTACTGGGGCCAGGGAACCCTGGTCAC VTVSSCGTCTCCTCAG T23 SEQ ID SEQ ID SEQ ID SEQ ID NO: 117 SEQ ID NO: 118NO: 114 NO: 115 NO: 116 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAQVQLQESGPGLVKPSETL GGSISRY IYYSGTT ARGLGDFGACCCTGTCCCTCACCTGCAGTGTCTCTGGTGGCTCCATTAGTAGAT SLTCSVSGGSISRYYWSW YATTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGG IRQPPGKGLEWIGYIYYSATTGGATATATCTATTACAGTGGGACCACCTACTATAACCCCTCCCT GTTYYNPSLKSRVTFSVDCAAGAGTCGAGTCACCTTTTCAGTAGACACGTCCAAGACCCAGTTCT TSKTQFSLKLNSVTAADTCCCTGAAACTTAACTCTGTGACCGCTGCGGACACGGCCGTATATTAC AVYYCARGLGDFWGRGTLTGTGCGAGAGGACTGGGGGACTTCTGGGGCCGGGGAACCCTGGTCAC VTVSS CGTCTCCTCAG T25SEQ ID SEQ ID SEQ ID SEQ ID NO: 125 SEQ ID NO: 126 NO: 122 NO: 123NO: 124 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCAGAQVQLQESGPGLVKPSETL GGSISSG INNSGNT ARGGSGDGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTG SLTCTVSGGSISSGIYYW IYY YGTATATACTACTGGAGTTGGATCCGCCAGCACCCAGGGAAGGGCCTG SWIRQHPGKGLEWIGYINGAGTGGATTGGATACATCAATAACAGTGGGAACACCTACTACAACCC NSGNTYYNPSLKGRVNISGTCCCTCAAGGGTCGAGTTAACATATCAGTAGACACGTCTAAGAAAC VDTSKKQFSLKLSSVTDAAGTTCTCCCTGAAGCTGAGCTCTGTGACTGACGCGGACACGGCCGTC DTAVYYCARGGSGDYWGQTATTACTGTGCGAGGGGGGGATCGGGCGACTACTGGGGCCAGGGAAC GTLVTVSSCCTGGTCACCGTCTCCTCAG

TABLE S-10B Anti-FX VL domain sequences and CDRs VL amino Ab VL LCDR1LCDR2 LCDR3 VL nucleotide sequence acid sequence T02 SEQ ID SEQ IDSEQ ID SEQ ID NO: 65 SEQ ID NO: 66 NO: 62 NO: 63 NO: 64CAGTCTGTCCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGC QSVLTQPPSASGTPGQRVTSSNIGSN RNT ATWDDSL AGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGISCSGSSSNIGSNYVYWYQ Y SAYVTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAA QLPGTAPKLLIYRNTQRPSCTCCTCATCTATAGGAATACTCAGCGGCCCTCAGAGGTCCCTGACC EVPDRFSGSKSGASASLAIGATTCTCTGGCTCCAAGTCTGGCGCCTCAGCCTCCCTGGCCATCAG SGLRSEDETDYYCATWDDSTGGGCTCCGGTCCGAGGATGAGACTGATTATTACTGTGCAACATGG LSAYVFGTGTKVTVLGATGACAGCCTGAGTGCTTATGTCTTCGGAACTGGGACCAAAGTCA CCGTCCTAG T05 SEQ IDSEQ ID SEQ ID SEQ ID NO: 75 SEQ ID NO: 76 NO: 72 NO: 73 NO: 74CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGAC QSALTQPPSASGSPGQSVTSSDVGGY EVN SSYAGSN AGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGISCTGTSSDVGGYYYVSWY YY TWVTTATTACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCC QQHPGKAPKLMIYEVNKRPAAACTCATGATTTATGAGGTCAATAAGCGGCCCTCAGGGGTCCCTG SGVPDRFSGSKSGITASLTATCGCTTCTCTGGCTCCAAGTCTGGCATCACGGCCTCCCTGACCGT VSGLQSEDEADYYCSSYAGCTCTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCAGCTCA SNTWVFGGGTKLTVLTATGCAGGCAGCAACACTTGGGTGTTCGGCGGAGGGACCAAGCTGA CCGTCCTAG T06 SEQ IDSEQ ID SEQ ID SEQ ID NO: 84 SEQ ID NO: 85 NO: 62 NO: 82 NO: 83CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGC QSVLTQPPSVSGTPGQRVTSSNIGSN RNN FGAGTKV AGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGISCSGSSSNIGSNYVYWYQ Y TVL TAATTATGTATACTGGTACCAGCAGTTCCCAGGAACGGCCCCCAAAQFPGTAPKLLIYRNNQRPS CTCCTCATCTATAGGAATAATCAGCGGCCCTCAGAGGTCCCTGACCEVPDRFSGSKSGASASLAI GATTCTCTGGCTCCAAGTCTGGCGCCTCAGCCTCCCTGGCCATCAGSGLRSEDETDYYCATWDDS TGGGCTCCGGTCCGAGGATGAGACTGATTATTACTGTGCAACATGGLSAYVFGAGTKVTVL GATGACAGCCTGAGTGCTTATGTCTTCGGAGCTGGGACCAAAGTCA CCGTCCTAGT12 SEQ ID SEQ ID SEQ ID SEQ ID NO: 94 SEQ ID NO: 95 NO: 91 NO: 92NO: 93 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGTATCTGTAGDIQMTQSPSSLSVSVGDRV QDISNY DAS QQYDNLPGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAA TITCQASQDISNYLNWYQQ ITCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC KPGKAPKLLIYDASNLETGCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGT VPSRFSGSGSGTDFTFIISTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCATCATCAGCAG SLQPEDIATYYCQQYDNLPCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGAT ITFGQGTRLEIKAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC T14 SEQ ID SEQ ID SEQ IDSEQ ID NO: 103 SEQ ID NO: 104 NO: 101 NO: 92 NO: 102GAAATTGTGTTGGCACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG EIVLAQSPATLSLSPGERAQSVNSY DAS QQRNNWP GGGAAAGAGCCACGTTCTCCTGCAGGGCCAGTCAGAGTGTTAACAGTFSCRASQSVNSYLAWHQQ IT CTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCKPGQAPRLLIYDASNRATG CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTIPARFSGSGSGTDFTLTIS TCAGTGGCAGTGGGTCCGGGACAGACTTCACTCTCACCATCAGCAGSLEPEDFAVYYCQQRNNWP CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAACITFGQGTRLEIK AACTGGCCTATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC T15 SEQ IDSEQ ID SEQ ID SEQ ID NO: 112 SEQ ID NO: 113 NO: 110 NO: 92 NO: 111GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG EIVLTQSPATLSLSPGERAQSVSSY DAS QQRSNWP GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTLSCRASQSVSSYLAWHQQ LT CTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCKPGQAPRLLIYDASNRATG CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTIPARFSGSGSGTDFTLTIS TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGSLEPEDFAVYYCQQRSNWP CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTAGCLTFGGGTKVEIK AACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC T23 SEQ IDSEQ ID SEQ ID SEQ ID NO: 120 SEQ ID NO: 121 NO: 119 NO: 92 NO: 111GAAATTGTGTTGACTCAGTCTCCAGCCACCCTGTCATTGTCTCCAG EIVLTQSPATLSLSPGERAQSVSGY DAS QQRSNWP GGGAAAGGGCCACCCTCTCCTGCCGGGCCAGTCAGAGTGTTAGCGGTLSCRASQSVSGYLAWHQQ LT CTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCKPGQAPRLLIYDASNRATG CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGATIPARFSGSGSGTDFTLTIS TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGSLEPEDFAVYYCQQRSNWP CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTAGCLTFGGGTKVEIK AACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC T25 SEQ IDSEQ ID SEQ ID SEQ ID NO: 130 SEQ ID NO: 131 NO: 128 NO: 92 NO: 129GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG EIVLTQSPATLSLSPGERAQSINNY DAS QQRNNWP GGGAAAGAGCCACCCTCTCCTGCAGGACCAGTCAGAGTATTAACAATLSCRTSQSINNYLAWFQQ PT CTACTTAGCCTGGTTCCAACAGAAACCTGGCCAGGCTCCCAGGCTCKPGQAPRLLIYDASNRAPG CTCATCTATGATGCATCCAACAGGGCCCCTGGCATCCCAGCCAGGTIPARFSGSGSGTDFTLTIS TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGSLEPEDFVVYFCQQRNNWP CCTGGAGCCTGAAGATTTTGTAGTTTATTTCTGTCAGCAGCGTAACPTFGQGTKVEIK AACTGGCCTCCGACATTCGGCCAAGGGACCAAGGTGGAAATCAAAC

TABLE S-10C Anti-FX VH domain sequences and CDRsThe following TxxxxH VH domains are suitable for pairing with a commonlight chain VL such as VL domain 0128L or 0325L. VH amino Ab VH HCDR1HCDR2 HCDR3 VH nucleotide sequence acid sequence T0200H SEQ ID SEQ IDSEQ ID SEQ ID NO: 465 SEQ ID NO: 466 NO: 462 NO: 463 NO: 464CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAAAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGRYSFTSY INPKTGD ARDGYGSGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATCTGCATT ASVKVSCKASRYSFT YT SARCLQL GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTSYYLHWVRQAPGQGL AAAACTGGTGACACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACEWMGIINPKTGDTSY CAGGGACACGTCCACGACCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAQKFQGRVTMTRDTS AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGGCCCGGTTTVYMELSSLRSED TGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCATAVYYCARDGYGSSA RCLQLWGQGTLVTVS S T0201H SEQ ID SEQ ID SEQ IDSEQ ID NO: 469 SEQ ID NO: 470 NO: 462 NO: 467 NO: 468CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPGINPKSGS ARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTASVKVSCKASRYSFT T SSRCLQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0202H SEQ ID SEQ ID SEQ ID SEQ ID NO: 471SEQ ID NO: 472 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGATACAGTCTGGGGCTGAGGTGCAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVQKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0203H SEQ ID SEQ ID SEQ ID SEQ ID NO: 473SEQ ID NO: 474 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGATCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELISLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0204H SEQ ID SEQ ID SEQ ID SEQ ID NO: 475SEQ ID NO: 476 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGCAGAAGACTGGGGCCTCAGT QVQLVQSGAEVQKTGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0205H SEQ ID SEQ ID SEQ ID SEQ ID NO: 478SEQ ID NO: 479 NO: 462 NO: 477 NO: 464CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGINPKSGD GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTASVKVSCKASRYSFT T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTSYYMHWVRQAPGQGL AAAAGTGGTGACACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACEWMGIINPKSGDTSY CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAACAGCCTGAGATCTGAQKFQGRVTMTRDTS AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGGCCCGGTSTVYMELNSLRSED TGCCTCCAGCTCTGGGGCCAGGGCGCCCTGGTCACCGTCTCCTCATAVYYCARDGYGSSA RCLQLWGQGTLVTVS S T0206H SEQ ID SEQ ID SEQ IDSEQ ID NO: 480 SEQ ID NO: 481 NO: 462 NO: 477 NO: 464CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTGACACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGDTSYCAGGGACACGTCCACGAGCACAGTCTACATGGACCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGGCCCGG TSTVYMDLSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSARCLQLWGQGTLVTVS S T0207H SEQ ID SEQ ID SEQ ID SEQ ID NO: 482SEQ ID NO: 483 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGACTGGGGCCTCAGT QVQLVQSGAEVKKTGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0208H SEQ ID SEQ ID SEQ ID SEQ ID NO: 484SEQ ID NO: 485 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGAACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG EQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0209H SEQ ID SEQ ID SEQ ID SEQ ID NO: 486SEQ ID NO: 487 NO: 462 NO: 463 NO: 464CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGCAAAAGCCTGGGGCCTCAGT QVQLVQSGAEVQKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATCTGCATT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYLHWVRQAPGQGLAAAACTGGTGACACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKTGDTSYCAGGGACACGTCCACGACCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGGCCCGG TTTVYMELSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSARCLQLWGQGTLVTVS S T0210H SEQ ID SEQ ID SEQ ID SEQ ID NO: 488SEQ ID NO: 499 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAACAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMELNSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0211H SEQ ID SEQ ID SEQ ID SEQ ID NO: 500SEQ ID NO: 501 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT ASVKVSCKASRYSFTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT SYYMHWVRQAPGQGLAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC EWMGIINPKSGSTSYCAGGGACACGTCCACGAGCACAGTCTACATGGACCTGAGCAGCCTGAGATCTG AQKFQGRVTMTRDTSAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TSTVYMDLSSLRSEDTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA TAVYYCARDGYGSSSRCLQLWGQGTLVTVS S T0212H SEQ ID SEQ ID SEQ ID SEQ ID NO: 505SEQ ID NO: 506 NO: 502 NO: 503 NO: 504CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGGFSFTSY INPRSGS ARDGYGSGAAGGTTTCCTGCAAGGCATCTGGATTCTCCTTCACCAGCTACTATATACACT ASVKVSCKASGFSFT YT SSRCFQY GGGTGCGCCAGGCCCCTGGACAAGGACTTGAGTGGATGGGAATAATCAACCCTSYYIHWVRQAPGQGL AGAAGTGGTAGCACAAGCTACGCTCAGAAGTTCCAGGGCAGAGTCACCATGACEWMGIINPRSGSTSY CAGGGACACGTCCACGAACACAGTCTACATGGACCTGAGCAGCCTGAGATCTGAQKFQGRVTMTRDTS AGGACACGGCCGTATATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGATNTVYMDLSSLRSED TGCTTCCAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCATAVYYCARDGYGSSS RCFQYWGQGTLVTVS S T0213H SEQ ID SEQ ID SEQ IDSEQ ID NO: 508 SEQ ID NO: 509 NO: 462 NO: 507 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGACTGGGGCCTCAGT QVQLVQSGAEVKKTGINPKSGT GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTASVKVSCKASRYSFT T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTSYYMHWVRQAPGQGL AAAAGTGGTACTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACEWMGIINPKSGTTSY CAGGGACACGTCCACGAGCACAGTCTACATGGAACTGAGCAGCCTGAGATCTGAQKFQGRVTMTRDTS AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGGTSTVYMELSSLRSED TGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCATAVYYCARDGYGSSS RCLQLWGQGTLVTVS S T0214H SEQ ID SEQ ID SEQ IDSEQ ID NO: 510 SEQ ID NO: 511 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGACTGGGGCCTCAGT QVQLVQSGAEVKKTGAAGGTTTCCTGCCAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT GASVKVSCQASRYSGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRCLQLWG QGTLVTVSS T0215H SEQ ID SEQ ID SEQ ID SEQ ID NO: 512SEQ ID NO: 513 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKPGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT GASVKVSCKASRYSGGGTGCGACAGGCCCCGGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRCLQLWG QGTLVTVSS T0216H SEQ ID SEQ ID SEQ ID SEQ ID NO: 514SEQ ID NO: 515 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLVQSGAEVKKTGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATTTGCACT GASVKVSCQASRYSGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRCLQLWG QGTLVTVSS T0217H SEQ ID SEQ ID SEQ ID SEQ ID NO: 516SEQ ID NO: 517 NO: 462 NO: 467 NO: 468CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGACGAAGCCTGGGGCCTCAGT QVQLVQSGAEVTKPGAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACT GASVKVSCKASRYSGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSTGCCTCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRCLQLWG QGTLVTVSS T0666H SEQ ID SEQ ID SEQ ID SEQ ID NO: 521SEQ ID NO: 522 NO: 462 NO: 467 NO: 520CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRIIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSATCATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRIIQLWG QGTLVTVSS T0667H SEQ ID SEQ ID SEQ ID SEQ ID NO: 524SEQ ID NO: 525 NO: 462 NO: 467 NO: 523CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRLIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRLIQLWG QGTLVTVSS T0668H SEQ ID SEQ ID SEQ ID SEQ ID NO: 527SEQ ID NO: 528 NO: 462 NO: 467 NO: 526CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRQIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCAGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRQIQLWG QGTLVTVSS T0669H SEQ ID SEQ ID SEQ ID SEQ ID NO: 530SEQ ID NO: 531 NO: 462 NO: 467 NO: 529CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRILMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSATCCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRILMLWG QGTLVTVSS T0670H SEQ ID SEQ ID SEQ ID SEQ ID NO: 533SEQ ID NO: 534 NO: 462 NO: 467 NO: 532CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRLLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCTGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRLLMLWG QGTLVTVSS T0671H SEQ ID SEQ ID SEQ ID SEQ ID NO: 536SEQ ID NO: 537 NO: 462 NO: 467 NO: 535CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRQLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCAGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRQLMLWG QGTLVTVSS T0672H SEQ ID SEQ ID SEQ ID SEQ ID NO: 539SEQ ID NO: 540 NO: 462 NO: 467 NO: 538CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRIIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSATCATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRIIMLWG QGTLVTVSS T0673H SEQ ID SEQ ID SEQ ID SEQ ID NO: 542SEQ ID NO: 543 NO: 462 NO: 467 NO: 541CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRLIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCTGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRLIMLWG QGTLVTVSS T0674H SEQ ID SEQ ID SEQ ID SEQ ID NO: 545SEQ ID NO: 546 NO: 462 NO: 467 NO: 544CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRQIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSCAGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRQIMLWG QGTLVTVSS T0675H SEQ ID SEQ ID SEQ ID SEQ ID NO: 548SEQ ID NO: 549 NO: 462 NO: 467 NO: 547CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRVIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSGTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRVIQLWG QGTLVTVSS T0676H SEQ ID SEQ ID SEQ ID SEQ ID NO: 551SEQ ID NO: 552 NO: 462 NO: 467 NO: 550CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRVLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSGTGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRVLMLWG QGTLVTVSS T0677H SEQ ID SEQ ID SEQ ID SEQ ID NO: 554SEQ ID NO: 555 NO: 462 NO: 467 NO: 553CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS SSRVIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTCGTCCCGG TRDTSTSTVYMELSGTGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSSSRVIMLWG QGTLVTVSS T0678H SEQ ID SEQ ID SEQ ID SEQ ID NO: 557SEQ ID NO: 558 NO: 462 NO: 467 NO: 556CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRIIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSATCATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRIIQLWG QGTLVTVSS T0679H SEQ ID SEQ ID SEQ ID SEQ ID NO: 560SEQ ID NO: 561 NO: 462 NO: 467 NO: 559CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRILMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSATCCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRILMLWG QGTLVTVSS T0680H SEQ ID SEQ ID SEQ ID SEQ ID NO: 563SEQ ID NO: 564 NO: 462 NO: 467 NO: 562CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRIIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSATCATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRIIMLWG QGTLVTVSS T0681H SEQ ID SEQ ID SEQ ID SEQ ID NO: 566SEQ ID NO: 567 NO: 462 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRLIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRLIQLWG QGTLVTVSS T0682H SEQ ID SEQ ID SEQ ID SEQ ID NO: 569SEQ ID NO: 570 NO: 462 NO: 467 NO: 568CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRLLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCTGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRLLMLWG QGTLVTVSS T0683H SEQ ID SEQ ID SEQ ID SEQ ID NO: 572SEQ ID NO: 573 NO: 462 NO: 467 NO: 571CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRLIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCTGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRLIMLWG QGTLVTVSS T0684H SEQ ID SEQ ID SEQ ID SEQ ID NO: 575SEQ ID NO: 576 NO: 462 NO: 467 NO: 574CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRQIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCAGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRQIQLWG QGTLVTVSS T0685H SEQ ID SEQ ID SEQ ID SEQ ID NO: 578SEQ ID NO: 579 NO: 462 NO: 467 NO: 577CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRQLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCAGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRQLMLWG QGTLVTVSS T0686H SEQ ID SEQ ID SEQ ID SEQ ID NO: 581SEQ ID NO: 582 NO: 462 NO: 467 NO: 580CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRQIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSCAGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRQIMLWG QGTLVTVSS T0687H SEQ ID SEQ ID SEQ ID SEQ ID NO: 584SEQ ID NO: 585 NO: 462 NO: 467 NO: 583CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRVIQLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSGTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRVIQLWG QGTLVTVSS T0688H SEQ ID SEQ ID SEQ ID SEQ ID NO: 587SEQ ID NO: 588 NO: 462 NO: 467 NO: 586CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRVLMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSGTGCTCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRVLMLWG QGTLVTVSS T0689H SEQ ID SEQ ID SEQ ID SEQ ID NO: 590SEQ ID NO: 591 NO: 462 NO: 467 NO: 589CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPARDGYGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS FSRVIMLGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCT FTSYYMHWVRQAPGAAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGAC QGLEWMGIINPKSGCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTG STSYAQKFQGRVTMAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGG TRDTSTSTVYMELSGTGATCATGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA SLRSEDTAVYYCARDGYGSFSRVIMLWG QGTLVTVSS T0713H SEQ ID SEQ ID SEQ ID SEQ ID NO: 593SEQ ID NO: 594 NO: 592 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRFSFTSY GAAGGTTTCCTGCAAGGCATCTAGATTCAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRFS Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0734H SEQ ID SEQ ID SEQ IDSEQ ID NO: 596 SEQ ID NO: 597 NO: 595 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRYHFTSY GAAGGTTTCCTGCAAGGCATCTAGATACCACTTCACCAGCTACTATATGCACTGASVKVSCKASRYH Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0736H SEQ ID SEQ ID SEQ IDSEQ ID NO: 599 SEQ ID NO: 600 NO: 598 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRYKFTSY GAAGGTTTCCTGCAAGGCATCTAGATACAAGTTCACCAGCTACTATATGCACTGASVKVSCKASRYK Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0742H SEQ ID SEQ ID SEQ IDSEQ ID NO: 602 SEQ ID NO: 603 NO: 601 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRYRFTSY GAAGGTTTCCTGCAAGGCATCTAGATACAGATTCACCAGCTACTATATGCACTGASVKVSCKASRYR Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0774H SEQ ID SEQ ID SEQ IDSEQ ID NO: 605 SEQ ID NO: 606 NO: 604 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRYSFKSY GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCAAGAGCTACTATATGCACTGASVKVSCKASRYS Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFKSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0785H SEQ ID SEQ ID SEQ IDSEQ ID NO: 608 SEQ ID NO: 609 NO: 607 NO: 467 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPRYSFTAY GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCGCCTACTATATGCACTGASVKVSCKASRYS Y GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTAYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0850H SEQ ID SEQ ID SEQ IDSEQ ID NO: 611 SEQ ID NO: 612 NO: 462 NO: 610 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPLNPKSGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATACTGAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGILNPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0925H SEQ ID SEQ ID SEQ IDSEQ ID NO: 614 SEQ ID NO: 615 NO: 462 NO: 613 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKIGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAATCGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKIG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0926H SEQ ID SEQ ID SEQ IDSEQ ID NO: 617 SEQ ID NO: 618 NO: 462 NO: 616 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKKGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAAGGGTAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKKG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0951H SEQ ID SEQ ID SEQ IDSEQ ID NO: 620 SEQ ID NO: 621 NO: 462 NO: 619 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKSSS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTAGCAGTACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSS CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0958H SEQ ID SEQ ID SEQ IDSEQ ID NO: 623 SEQ ID NO: 624 NO: 462 NO: 622 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKSGD GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS T GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTGACACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGDTSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0989H SEQ ID SEQ ID SEQ IDSEQ ID NO: 626 SEQ ID NO: 627 NO: 462 NO: 625 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKSGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS R GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTAGAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSRSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0990H SEQ ID SEQ ID SEQ IDSEQ ID NO: 629 SEQ ID NO: 630 NO: 462 NO: 628 NO: 565CAGGTGCAGTTGATACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGT QVQLIQSGAEVKKPINPKSGS GAAGGTTTCCTGCAAGGCATCTAGATACAGCTTCACCAGCTACTATATGCACTGASVKVSCKASRYS S GGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTFTSYYMHWVRQAPG AAAAGTGGTAGTAGCAGTTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACQGLEWMGIINPKSG CAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGSSSYAQKFQGRVTM AGGACACGGCCGTGTATTACTGTGCGAGAGATGGGTATGGCAGCTTCTCCCGGTRDTSTSTVYMELS CTGATCCAGCTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASLRSEDTAVYYCAR DGYGSFSRLIQLWG QGTLVTVSS T0999H SEQ ID SEQ ID SEQ IDSEQ ID NO: 631 SEQ ID NO: 632 NO: 598 NO: 467 NO: 565CAGGTTCAGCTGATTCAGTCCGGCGCCAAAGTGAAGAAACCTGGCGCCTCTGT QVQLIQSGAKVKKPGAAGGTGTCCTGCAAGGCCTCTCGGTACAAGTTCACCTCCTACTACATGCACT GASVKVSCKASRYKGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATCATCAACCCC FTSYYMHWVRQAPGAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGAC QGLEWMGIINPKSGCAGAGACACCTCTACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCG STSYAQKFQGRVTMAGGACACCGCCGTGTACTACTGTGCCAGAGATGGCTACGGCAGCTTCTCCAGA TRDTSTSTVYMELSCTGATCCAGTTGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCT SLRSEDTAVYYCARDGYGSFSRLIQLWG QGTLVTVSS SEQ ID NO: 636. Consensus HCDR1. RYXFTSYYX is K or SRepresenting the T0201H VH CDR1 RYSFTSYY (SEQ ID NO: 462) in which the Ser at IMGTposition 29 is retained or replaced by Lys.SEQ ID NO: 637. Consensus HCDR3. ARDGYGSX₁SRX₂X₃QLX1 is F or S. X2 is any amino acid. X3 is I or L.Representing the T0201H CDR3 ARDGYGSSSRCLQL (SEQ ID NO: 468) in which theSer at IMGT position 111A is retained or replaced by Phe, the Cys at IMGTposition 114 is retained or replaced by another amino acid residue, and theLeu at IMGT position 115 is retained or replaced by Ile.SEQ ID NO: 638. Consensus HCDR3. ARDGYGSX₁SRX₂X₃QLX1 is F or S. X2 is Leu or Val. X3 is I or L.Representing the T0201H CDR3 ARDGYGSSSRCLQL (SEQ ID NO: 468) in which theSer at IMGT position 111A is retained or replaced by Phe, the Cys at IMGTposition 114 is replaced by Leu or Val, and the Leu at IMGT position 115 isretained or replaced by Ile. SEQ ID NO: 639. Consensus HCDR3.ARDGYGSFSRXIQL X is Leu or Val.Representing the T0201H CDR3 ARDGYGSSSRCLQL (SEQ ID NO: 468) in which theSer at IMGT position 111A is replaced by Phe, the Cys at IMGT position 114is replaced by Leu or Val, and the Leu at IMGT position is replaced by Ile.

TABLE S-11 Heavy chain sequences SEQ ID Nucleic acidGAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCCNO: 418 encodingGGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAACN1280H-IgG4-ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCCP K439E;AAGAAATCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGCN1280 codingTACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACCsequenceAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGunderlined.GTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCTSEQ ID N1280H-IgG4-EVQLVESGGGFVQPGGSLRLSCAVSGFRFNSYWMSWVRQAPGKGLEWVANINQDGSRKFYVASVKGRFTMSRDNANO: 419 P K439EKKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLamino acidVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGsequencePPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSPSEQ ID Nucleic acidCAGGTTCAGCTGATTCAGTCCGGCGCCAAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCTNO: 420 encodingCGGTACAAGTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATCT0999H-IgG4-ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCTP E356K;ACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGCT0999H codingTACGGCAGCTTCTCCAGACTGATCCAGTTGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCTGCTTCCACCAAGsequenceGGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCunderlinedAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ IDT0999H-IgG4-QVQLIQSGAKVKKPGASVKVSCKASRYKFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTSNO: 421 P E356KTSTVYMELSSLRSEDTAVYYCARDGYGSFSRLIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVamino acidKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPsequencePCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPSEQ ID Nucleic acidGAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCCNO: 423 encodingGGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAACN1454H-IgG4-ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCCP K439EAAGAAAGAGGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGCTACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCTSEQ ID N1454H-IgG4-EVQLVESGGGFVQPGGSLRLSCAVSGFRFNSYWMSWVRQAPGKGLEWVANINQDGSRKFYVASVKGRFTMSRDNANO: 424 P K439EKKEVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLamino acidVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGsequencePPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSPSEQ ID Nucleic acidGAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCCNO: 425 encodingGGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAACN1441H-IgG4-ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCCP K439EGACAAGTCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGCTACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCTSEQ ID N1441H-IgG4-EVQLVESGGGFVQPGGSLRLSCAVSGFRFNSYWMSWVRQAPGKGLEWVANINQDGSRKFYVASVKGRFTMSRDNANO: 426 P K439EDKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLamino acidVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGsequencePPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSPSEQ ID Nucleic acidGAAGTGCAGCTGGTTGAATCTGGCGGCGGATTTGTTCAGCCTGGCGGCTCTCTGAGACTGTCCTGTGCTGTGTCCNO: 427 encodingGGCTTCCGGTTCAACTCCTACTGGATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCAACN1442H-IgG4-ATCAACCAGGACGGCTCCCGGAAGTTCTACGTGGCCTCTGTGAAGGGCAGATTCACCATGTCTCGGGACAACGCCP K439EGAGAAGTCCGTGTACGTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCTAGAGAGGGCTACTCCTCCATCAAGTACTACGGCATGGACGTGTGGGGCCAGGGCACAACCGTGACAGTCTCTTCCGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCTSEQ ID N1442H-IgG4-EVQLVESGGGFVQPGGSLRLSCAVSGFRFNSYWMSWVRQAPGKGLEWVANINQDGSRKFYVASVKGRFTMSRDNANO: 428 P K439EEKSVYVQMNSLRAEDTAVYYCAREGYSSIKYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLamino acidVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGsequencePPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSPSEQ ID Nucleic acidCAGGTTCAGCTGATTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCTNO: 429 encodingCGGTACAAGTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATCT0736H-IgG4-ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCTP E356KACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGCTACGGCAGCTTCTCCAGGCTGATCCAGTTGTGGGGACAGGGCACACTGGTCACCGTGTCCTCTGCTTCTACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ IDT0736H-IgG4-QVQLIQSGAEVKKPGASVKVSCKASRYKFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTSNO: 430 P E356KTSTVYMELSSLRSEDTAVYYCARDGYGSFSRLIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVamino acidKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPsequencePCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPSEQ ID Nucleic acidCAGGTTCAGCTGATTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCCNO: 431 encodingAGATACTCCTTCACCTCCTACTACATGCACTGGGTCCGACAGGCCCCTGGACAAGGATTGGAGTGGATGGGCATCT0687H-IgG4-ATCAACCCCAAGTCCGGCTCCACCTCTTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCAGAGACACCTCTP E356KACCTCCACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCCAGAGATGGCTACGGCTCCTTCAGCAGAGTGATCCAGTTGTGGGGCCAGGGCACACTGGTCACAGTGTCCTCTGCTTCCACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT SEQ IDT0687H-IgG4-QVQLIQSGAEVKKPGASVKVSCKASRYSFTSYYMHWVRQAPGQGLEWMGIINPKSGSTSYAQKFQGRVTMTRDTSNO: 432 P E356KTSTVYMELSSLRSEDTAVYYCARDGYGSFSRVIQLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVamino acidKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPsequencePCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP

Human Germline Gene Segments

TABLE S-12 Corresponding qermline v and j gene segments for antibody VHand VL domains V -- J Anti-FIX heavy chain N128 IGHV3-7*01--IGHJ6*02N183 IGHV3-48*02--IGHJ6*02 Anti FX heavy chain T0200IGHV1-46*03--IGHJ1*01 Common light chain N0128L IGLV3-21*d01--IGOLJ2*01

Common Light Chain Sequences

TABLE S-50A N0128 and N0325 VL domain sequences and CDRs VL amino acidAb VL LCDR1 LCDR2 LCDR3 VL nucleotide sequence sequence N128L SEQ IDSEQ ID SEQ ID SEQ ID NO: 9 SEQ ID NO: 10 NO: 6 NO: 7 NO: 8TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAGA SYVLTQPPSVSVAPGETANIGRKS YDS QVWDGS GACGGCCAGGATTACCTGTGGGGGAGACAACATTGGAAGGAAAAGTGRITCGGDNIGRKSVYWYQ SDHWV TGTACTGGTACCAGCAGAAGTCAGGCCAGGCCCCTGTGCTGGTCATCQKSGQAPVLVIYYDSDRP TATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGSGIPERFSGSNSGNTATL GTCCAACTCTGGGAACACGGCGACCCTGACCATCAGCAGGGTCGAAGTISRVEAGDEADYYCQVW CCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATGGAAGTAGTDGSSDHWVFGGGTKLTVL GATCATTGGGTGTTCGGCGGAGGGACCAAGTTGACCGTCCTAG N325LSEQ ID SEQ ID SEQ ID SEQ ID NO: 415 SEQ ID NO: 416 NO: 6 NO: 7 NO: 8TACGTGCTGACCCAGCCTCCTTCCGTGTCTGTTGCTCCTGGCGAGAC YVLTQPPSVSVAPGETARAGCCAGAATCACCTGTGGCGGCGATAACATCGGCCGGAAGTCCGTGT ITCGGDNIGRKSVYWYQQACTGGTATCAGCAGAAGTCCGGCCAGGCTCCTGTGCTGGTCATCTAC KSGQAPVLVIYYDSDRPSTACGACTCCGACCGGCCTTCTGGCATCCCTGAGAGATTCTCCGGCTC GIPERFSGSNSGNTATLTCAACTCCGGCAATACCGCCACACTGACCATCTCCAGAGTGGAAGCTG ISRVEAGDEADYYCQVWDGCGACGAGGCCGACTACTACTGCCAAGTGTGGGACGGCTCCTCTGAC GSSDHWVFGGGTKLTVLCACTGGGTTTTCGGCGGAGGCACCAAGCTGACAGTGCTG

TABLE S-50B N0128 and N0325 VL domain framework sequences Ab VL FR1 FR2FR3 FR4 N128L SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO: 138SEQ ID NO: 139 SYVLTQPPSVSVAP VYWYQQKSGQAPVL DRPSGIPERFSGSN FGGGTKLTVLGETARITCGGD VIY SGNTATLTISRVEA GDEADYYC N325L SEQ ID NO: 417SEQ ID NO: 137 SEQ ID NO: 138 SEQ ID NO: 139 YVLTQPPSVSVAPG ETARITCGGD

TABLE S-50C N0128 and N0325 light chain sequences SEQ IDN0128L-IgL CodingTCCTATGTGC TGACTCAGCC ACCCTCAGTG TCAGTGGCCC CAGGAGAGAC GGCCAGGATTNO: 404 nucleic acidACCTGTGGGG GAGACAACAT TGGAAGGAAA AGTGTGTACT GGTACCAGCA GAAGTCAGGCCAGGCCCCTG TGCTGGTCAT CTATTATGAT AGCGACCGGC CCTCAGGGAT CCCTGAGCGATTCTCTGGGT CCAACTCTGG GAACACGGCG ACCCTGACCA TCAGCAGGGT CGAAGCCGGGGATGAGGCCG ACTATTACTG TCAGGTGTGG GATGGAAGTA GTGATCATTG GGTGTTCGGCGGAGGGACCA AGTTGACCGT CCTAGGTCAG CCCAAGGCTG CCCCCTCGGT CACTCTGTTCCCACCCTCCT CTGAGGAGCT TCAAGCCAAC AAGGCCACAC TGGTGTGTCT CATAAGTGACTTCTACCCGG GAGCCGTGAC AGTGGCCTGG AAGGCAGATA GCAGCCCCGT CAAGGCGGGAGTGGAGACCA CCACACCCTC CAAACAAAGC AACAACAAGT ACGCGGCCAG CAGCTACCTGAGCCTGACGC CTGAGCAGTG GAAGTCCCAC AAAAGCTACA GCTGCCAGGT CACGCATGAAGGGAGCACCG TGGAGAAGAC AGTGGCCCCT ACAGAATGTT CA SEQ ID N0128L light chainSYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLNO: 405 amino acid sequenceTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG(mature)AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECSSEQ ID N0325L-IgL codingTACGTGCTGACCCAGCCTCCTTCCGTGTCTGTTGCTCCTGGCGAGACAGCCAGAATCACCTGTGGCGGCGATNO: 413 nucleic acid; N0325LAACATCGGCCGGAAGTCCGTGTACTGGTATCAGCAGAAGTCCGGCCAGGCTCCTGTGCTGGTCATCTACTACcoding sequenceGACTCCGACCGGCCTTCTGGCATCCCTGAGAGATTCTCCGGCTCCAACTCCGGCAATACCGCCACACTGACCunderlinedATCTCCAGAGTGGAAGCTGGCGACGAGGCCGACTACTACTGCCAAGTGTGGGACGGCTCCTCTGACCACTGGGTTTTCGGCGGAGGCACCAAGCTGACAGTGCTGGGACAACCTAAGGCCGCTCCTTCTGTGACCCTGTTTCCTCCATCCTCCGAGGAACTGCAGGCCAACAAGGCTACCCTCGTGTGCCTGATCTCCGACTTTTACCCTGGCGCTGTGACCGTGGCCTGGAAGGCTGATAGTTCTCCTGTGAAGGCCGGCGTGGAAACCACCACACCTTCCAAGCAGTCCAACAACAAATACGCCGCCTCCTCCTACCTGTCTCTGACCCCTGAACAGTGGAAGTCCCACAAGTCCTACTCTTGCCAAGTGACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCTCCTACCGAGTGCTCC SEQ IDN0325L light chainYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTNO: 414 amino acid sequenceISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA(mature)VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

Recombinant expression of bispecific antibody using common light chainN0128L with its native human Igλ leader sequence (v3-21 leader peptideMAWTALLLGLLSHCTGSVT SEQ ID NO: 519) resulted in clipping of the Nterminal Ser to produce antibody in which the VL domain was identical tothe sequence shown herein for N0325 VL domain. For use with alternativeleader sequences in which the mature light chain polypeptide is producedby cleavage after the Ser, the light chain 0325 was generated in orderto achieve the same mature product. 0325 omits the N terminal Serresidue of 0128L.

Constant Regions

TABLE S-100 Antibody constant region sequences IgG4 PE humanSEQ ID NO: 143 heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPconstant regionSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4 human heavy SEQ ID NO: 144chain constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPregion withSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVknobs-into-holesDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTImutations andSKAKGQPREPQVCTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYShinge mutation. RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Type a (IgG4ra)IgG4 human heavy SEQ ID NO: 145 chain constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPregion withSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVknobs-into-holesDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTImutations andSKAKGQPREPQVYTLPPSQCEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVShinge mutation. RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Type b (IgG4yb)IgG4 human heavy SEQ ID NO: 409 chain constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPregion withSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVP (hinge) mutationDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIand K439ESKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP IgG4 human heavy SEQ ID NO: 410chain constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPregion withSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVP (hinge) mutationDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIand E356KSKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP IgG4-P K439E SEQ ID NO: 411encoding nucleicGCTTCCACCAAGGGACCCAGCGTTTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTacidCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCTAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAAGAGTCCCTGTCTCTGTCCCCT IgG4-P E356K SEQ ID NO: 412encoding nucleicGCTTCCACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCTTGCTCCAGATCCACCTCCGAGTCTACAGCTGCTacidCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCCAGACCTACACCTGTAATGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCTGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCTCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGAGAACCCCAGGTTTACACCCTGCCTCCAAGCCAGAAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCTCCTGTGCTGGACTCCGATGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAAGAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCT Nucleic acid SEQ ID NO: 633encoding IgLGGACAACCTAAGGCCGCTCCTTCTGTGACCCTGTTTCCTCCATCCTCCGAGGAACTGCAGGCCAACAAGGCThuman lambdaACCCTCGTGTGCCTGATCTCCGACTTTTACCCTGGCGCTGTGACCGTGGCCTGGAAGGCTGATAGTTCTCCTlight chainGTGAAGGCCGGCGTGGAAACCACCACACCTTCCAAGCAGTCCAACAACAAATACGCCGCCTCCTCCTACCTGconstant regionTCTCTGACCCCTGAACAGTGGAAGTCCCACAAGTCCTACTCTTGCCAAGTGACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCTCCTACCGAGTGCTCC Human lambda SEQ ID NO: 146 light chainGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLconstant region SLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS Human kappaSEQ ID NO: 147 light chainKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTconstant region LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE N N128H Alanine Scanning Mutants CDR1 (GFTFNSW) G F T F N S Y W(SEQ ID NO: 1) N400H N401H N402H N403H N404H N405H N406H N407HCDR2 (INQDGSEK) I N Q D G S E K (SEQ ID NO: 2) N408H N409H N410H N411HN412H N413H N414H N415H CDR3 (ARE A R E G Y S S S S Y Y G M D VGYSSSSYYGMDV) N416H N417H N418H N419H N420H N421H N422H N423H N424HN425H N426H N427H N428H N429H (SEQ ID NO: 3) N128H CDR3 Mutants CDR3 A CD E F G H I K L M N P Q R S T V W Y AREGYSSSSYYGMDV N420H N467H N468HN469H N470H N471H N472H N473H N474H N475H N476H N477H N478H N479H N480HN128H N481H N482H N483H N484H (SEQ ID NO: 3) AREGYSSSSYYGMDV N421H N485HN486H N487H N488H N489H N490H N491H N492H N493H N494H N495H N496H N497HN498H N128H N499H N500H N501H N502H (SEQ ID NO: 3) AREGYSSSSYYGMDV N422HN430H N431H N432H N433H N434H N435H N436H N437H N438H N439H N440H N441HN442H N443H N128H N444H N445H N446H N447H (SEQ ID NO: 3) AREGYSSSSYYGMDVN423H N448H N449H N450H N451H N452H N453H N454H N455H N456H N457H N458HN459H N460H N461H N128H N462H N463H N464H N465H (SEQ ID NO: 3)AREGYSSSSYYGMDV N424H N542H N543H N544H N545H N546H N547H N548H N549HN550H N551H N552H N553H N554H N555H N559H N556H N557H N558H N128H(SEQ ID NO: 3) AREGYSSSSYYGDV N425H N561H N562H N563H N564H N565H N566HN567H N568H N569H N570H N571H N572H N573H N574H N578H N575H N576H N577HN128H (SEQ ID NO: 3) AREGYSSSSYYGMDV N426H N579H N580H N581H N582H N128HN583H N584H N585H N586H N587H N588H N589H N590H N591H N592H N593H N594HN595H N596H (SEQ ID NO: 3) AREGYSSSSYYGMDV N427H N597H N598H N599H N600HN601H N602H N603H N604H N605H N128H N606H N607H N608H N609H N610H N611HN612H N613H N614H (SEQ ID NO: 3) AREGYSSSSYYGMDV N428H N615H N128H N616HN617H N618H N619H N620H N621H N622H N623H N624H N625H N626H N627H N628HN629H N630H N631H N632H (SEQ ID NO: 3) AREGYSSSSYYGMDV N429H N633H N634HN635H N636H N637H N638H N639H N640H N641H N642H N643H N644H N645H N646HN647H N648H N128H N649H N650H (SEQ ID NO: 3) AREGYSSSSYYGMDV N128H N651HN652H N653H N654H N655H N656H N657H N658H N659H N660H N661H N662H N663HN664H N665H N666H N667H N668H N669H (SEQ ID NO: 3) AREGYSSSSYYGMDV N416HN670H N671H N672H N673H N674H N675H N676H N677H N678H N679H N680H N681HN682H N128H N683H N684H N685H N686H N687H (SEQ ID NO: 3) AREGYSSSSYYGMDVN417H N689H N690H N128H N691H N692H N693H N694H N695H N696H N697H N698HN699H N700H N701H N702H N703H N704H N705H N706H (SEQ ID NO: 3)AREGYSSSSYYGMDV N418H N707H N708H N709H N710H N128H N711H N712H N713HN714H N715H N716H N717H N718H N719H N720H N721H N722H N723H N724H(SEQ ID NO: 3) AREGYSSSSYYGMDV N419H N725H N726H N727H N728H N729H N730HN731H N732H N733H N734H N735H N736H N737H N738H N739H N740H N741H N742HN128H (SEQ ID NO: 3) N436H CDR3 Mutants CDR3 A C D E F G H I K L M N P QR S T V W Y AREGYSSISYYGMDV N503H N504H N505H N506H N507H N508H N509HN510H N511H N512H N513H N514H N515H N516H N517H N436H N518H N519H N520HN521H (SEQ ID NO: 171) AREGYSSISYYGMDV N522H N523H N524H N525H N526HN527H N528H N529H N530H N531H N532H N533H N534H N535H N536H N436H N537HN538H N539H N540H (SEQ ID NO: 171) N436H CDR1 Mutants CDR1 A C D E F G HI K L M N P Q R S T V W Y GFTFNSYW N819H N820H N821H N822H N436H N823HN824H N825H N826H N827H N828H N829H N830H N831H N832H N833H N834H N835HN836H (SEQ ID NO: 1) GFTFNSYW N837H N838H N839H N436H N840H N841H N842HN843H N844H N845H N846H N847H N848H N849H N850H N851H N852H N853H N854H(SEQ ID NO: 1) GFTFNSYW N856H N857H N858H N859H N860H N861H N862H N863HN864H N865H N866H N867H N868H N869H N870H N436H N871H N872H N873H(SEQ ID NO: 1) GFTFNSYW N874H N875H N876H N436H N877H N878H N879H N880HN881H N882H N883H N884H N885H N886H N887H N888H N889H N890H N891H(SEQ ID NO: 1) GFTFNSYW N892H N893H N894H N895H N896H N897H N898H N899HN900H N901H N436H N902H N903H N904H N905H N906H N907H N908H N909H(SEQ ID NO: 1) GFTFNSYW N910H N911H N912H N913H N914H N915H N916H N917HN918H N919H N920H N921H N922H N923H N436H N924H N925H N926H N927H(SEQ ID NO: 1) GFTFNSYW N928H N929H N930H N931H N932H N933H N934H N935HN936H N937H N938H N939H N940H N941H N942H N943H N944H N945H N436H(SEQ ID NO: 1) GFTFNSYW N946H N947H N948H N949H N950H N951H N952H N953HN954H N955H N956H N957H N958H N959H N960H N961H N962H N436H N963H(SEQ ID NO: 1) N436H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S TV W Y INQDGSEK N964H N965H N966H N967H N968H N969H N970H N436H N971HN972H N973H N974H N975H N976H N977H N978H N979H N980H N981H N982H(SEQ ID NO: 2) INQDGSEK N983H N984H N985H N986H N987H N988H N989H N990HN991H N992H N993H N436H N994H N995H N996H N997H N998H N999H N1000HN1001H (SEQ ID NO: 2) INQDGSEK N1002H N1003H N1004H N1005H N1006H N1007HN1008H N1009H N1010H N1011H N1012H N1013H N1014H N436H N1015H N1016HN1017H N1018H N1019H N1020H (SEQ ID NO: 2) INQDGSEK N1021H N1022H N436HN1023H N1024H N1025H N1026H N1027H N1028H N1029H N1030H N1031H N1032HN1033H N1034H N1035H N1036H N1037H N1038H N1039H (SEQ ID NO: 2) INQDGSEKN1040H N1041H N1042H N1043H N1044H N436H N1045H N1046H N1047H N1048HN1049H N1050H N1051H N1052H N1053H N1054H N1055H N1056H N1057H N1058H(SEQ ID NO: 2) INQDGSEK N1059H N1060H N1061H N1062H N1063H N1064H N1065HN1066H N1067H N1068H N1069H N1070H N1071H N1072H N1073H N436H N1074HN1075H N1076H N1077H (SEQ ID NO: 2) INQDGSEK N1078H N1079H N1080H N436HN1081H N1082H N1083H N1084H N1085H N1086H N1087H N1088H N1089H N1090HN1091H N1092H N1093H N1094H N1095H N1096H (SEQ ID NO: 2) INQDGSEK N1097HN1098H N1099H N1100H N1101H N1102H N1103H N1104H N436H N1105H N1106HN1107H N1108H N1109H N1110H N1111H N1112H N1113H N1114H N1115H(SEQ ID NO: 2) N511H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S TV W Y INQDGSEK N1116H N1117H N1118H (SEQ ID NO: 2) INQDGSEK(SEQ ID NO: 2) INQDGSEK N1119H (SEQ ID NO: 2) INQDGSEK N1120H N1121HN511H N1122H N1123H N1124H N1125H N1126H N1127H N1128H N1129H N1130HN1131H N1132H N1133H N1134H N1135H N1136H N1137H N1138H (SEQ ID NO: 2)INQDGSEK N1139H (SEQ ID NO: 2) INQDGSEK N1140H N1141H N1142H N1143HN1144H N1145H N1146H N1147H N1148H N1149H N1150H N1151H N1152H N1153HN1154H N511H N1155H N1156H N1157H N1158H (SEQ ID NO: 2) INQDGSEK N1159HN1160H N1161H N511H N1162H N1163H N1164H N1165H N1166H N1167H N1168HN1169H N1170H N1171H N1172H N1173H N1174H N1175H N1176H N1177H(SEQ ID NO: 2) INQDGSEK (SEQ ID NO: 2)Selected N436H CDR1 Mutants (batch 1) CDR1 A C D E F G H I K L M N P Q RS T V W Y GFTFNSYW N825H N832H N833H (SEQ ID NO: 1) GFTFNSYW N849H(SEQ ID NO: 1) GFTFNSYW N863H N866H (SEQ ID NO: 1) GFTFNSYW N875H N878HN889H (SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1) GFTFNSYW N917H N920H N921HN925H (SEQ ID NO: 1) GFTFNSYW N934H N936H N937H N939H N940H N941H N942HN943H N944H N945H (SEQ ID NO: 1) GFTFNSYW N946H N947H N948H N949H N950HN951H N952H N953H N954H N955H N956H N957H N963H (SEQ ID NO: 1)N511H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R S T V W Y GFTFNSYWN1178H N1179H N1180H (SEQ ID NO: 1) GFTFNSYW N1181H (SEQ ID NO: 1)GFTFNSYW N1182H N1183H (SEQ ID NO: 1) GFTFNSYW N1211H N1212H N1213H(SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1) GFTFNSYW N1184H N1185H N1186HN1187H (SEQ ID NO: 1) GFTFNSYW N1188H N1189H N1190H N1191H N1192H N1193HN1194H N1195H N1196H N1197H (SEQ ID NO: 1) GFTFNSYW N1198H N1199H N1200HN1201H N1202H N1203H N1204H N1205H N1206H N1207H N1208H N1209H N1210H(SEQ ID NO: 1) N1172H CDR1 Mutants CDR1 A C D E F G H I K L M N P Q R ST V W Y GFTFNSYW N1214H N1215H N1216H (SEQ ID NO: 1) GFTFNSYW N1217H(SEQ ID NO: 1) GFTFNSYW N1218H N1219H (SEQ ID NO: 1) GFTFNSYW N1247HN1248H N1249H (SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1) GFTFNSYW N1220HN1221H N1222H N1223H (SEQ ID NO: 1) GFTFNSYW N1224H N1225H N1226H N1227HN1228H N1229H N1230H N1231H N1232H N1233H (SEQ ID NO: 1) GFTFNSYW N1234HN1235H N1236H N1237H N1238H N1239H N1240H N1241H N1242H N1243H N1244HN1245H N1246H (SEQ ID NO: 1) Selected N436H CDR1 Mutants (batch 2) CDR1A C D E F G H I K L M N P Q R S T V W Y GFTFNSYW (SEQ ID NO: 1) GFTFNSYW(SEQ ID NO: 1) GFTFNSYW N869H N872H N873H (SEQ ID NO: 1) GFTFNSYW N877HN886H N888H N889H N891H (SEQ ID NO: 1) GFTFNSYW N892H N893H N894H N895HN896H N897H N898H N899H N900H N901H N902H N903H N904H N905H N906H N907HN908H N909H (SEQ ID NO: 1) GFTFNSYW N915H N923H N926H (SEQ ID NO: 1)GFTFNSYW N937H (SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1) N511H CDR1 MutantsA C D E F G H I K L M N P Q R S T V W Y GFTFNSYW (SEQ ID NO: 1) GFTFNSYW(SEQ ID NO: 1) GFTFNSYW N1250H N1251H N1252H (SEQ ID NO: 1) GFTFNSYWN1253H N1254H N1255H N1256H N1257H (SEQ ID NO: 1) GFTFNSYW N1258H N1259HN1260H N1261H N1262H N1263H N1264H N1265H N1266H N1267H N1268H N1269HN1270H N1271H N1272H N1273H N1274H N1275H (SEQ ID NO: 1) GFTFNSYW N1276HN1277H N1278H (SEQ ID NO: 1) GFTFNSYW N1279H (SEQ ID NO: 1) GFTFNSYW(SEQ ID NO: 1) N1172H CDR1 Mutants A C D E F G H I K L M N P Q R S T V WY GFTFNSYW (SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1) GFTFNSYW N1280H N1281HN1282H (SEQ ID NO: 1) GFTFNSYW N1283H N1284H N1285H N1286H N1287H(SEQ ID NO: 1) GFTFNSYW N1288H N1289H N1290H N1291H N1292H N1293H N1294HN1295H N1296H N1297H N1298H N1299H N1300H N1301H N1302H N1303H N1304HN1305H (SEQ ID NO: 1) GFTFNSYW N1306H N1307H N1308H (SEQ ID NO: 1)GFTFNSYW N1309H (SEQ ID NO: 1) GFTFNSYW (SEQ ID NO: 1)N1280H CDR2 Mutants CDR2 A C D E F G H I K L M N P Q R S T V W YINQDGSRK N1310H N1311H N1280H N1312H N1313H N1314H N1315H N1316H N1317HN1318H N1319H N1320H N1321H N1322H N1323H N1324H N1325H N1326H N1327HN1328H (SEQ ID NO: 436) INQDGSRK N1329H N1330H N1331H N1332H N1333HN1334H N1335H N1336H N1337H N1338H N1339H N1340H N1341H N1342H N1343HN1280H N1344H N1345H N1346H N1347H (SEQ ID NO: 436) N1280H CDR1 MutantsCDR1 A C D E F G H I K L M N P Q R S T V W Y GFRFNSYW N1367H N1368HN1369H N1370H N1371H N1372H N1373H N1374H N1375H N1376H N1377H N1378HN1379H N1380H N1381H N1382H N1383H N1384H N1385H N1280H (SEQ ID NO: 441)N1280H CDR1 Double Mutants (all including Arg29Lys, with Tyr37 mutated as shown)CDR1 A C D E F G H I K L M N P Q R S T V W Y GFKFNSYW N1348H N1349HN1360H N1351H N1352H N1353H N1354H N1355H N1356H N1357H N1358H N1359HN1360H N1361H N1362H N1363H N1364H N1365H N1366H (SEQ ID NO: 647)

TABLE T T0201H CDR3 Mutants CDR3 A C D E F G H I K L M N P Q R S T V W YARDGYGSSSRCLQL T400H T401H T402H T403H T404H T405H T406H T407H T408HT409H T410H T411H T412H T413H T414H T415H T416H T417H T418H(SEQ ID NO: 468) ARDGYGSSSRCLQL T419H T420H T421H T422H T423H T424HT425H T426H T427H T428H T429H T430H T431H T432H T433H T434H T435H T436HT437H (SEQ ID NO: 468) ARDGYGSSSRCLQL T438H T439H T440H T441H T442HT443H T444H T445H T446H T447H T448H T449H T450H T451H T452H T453H T454HT455H T456H (SEQ ID NO: 468) ARDGYGSSSRCLQL T457H T458H T459H T460HT461H T462H T463H T464H T465H T466H T467H T468H T469H T470H T471H T472HT473H T474H T475H (SEQ ID NO: 468) ARDGYGSSSRCLQL T476H T477H T478HT479H T480H T481H T482H T483H T484H T485H T486H T487H T488H T489H T490HT491H T492H T493H T494H (SEQ ID NO: 468) ARDGYGSSSRCLQL T495H T496HT497H T498H T499H T500H TSOIH T502H T503H T504H T505H T506H T507H T508HT509H T510H T511H T512H T513H (SEQ ID NO: 468) ARDGYGSSSRCLQL T514HTS15H T516H T517H T518H T519H TS20H T521H T522H T523H TS24H T525H T526HT527H T528H T529H T530H T531H T532H (SEQ ID NO: 468) ARDGYGSSSRCLQLT533H T534H T535H T536H T537H T538H T539H T540H T541H T542H T543H T544HT545H T546H T547H T548H T549H T550H T551H (SEQ ID NO: 468)ARDGYGSSSRCLQL T552H T553H T554H T555H T556H T557H T558H T559H T560HT561H T562H T563H T564H T565H T566H T567H T568H T569H T570H(SEQ ID NO: 468) ARDGYGSSSRCLQL T571H T572H T573H T574H T575H T576HT577H T578H T579H T580H T581H T582H T583H T584H T585H T586H T587H T588HT589H (SEQ ID NO: 468) ARDGYGSSSRCLQL T590H T591H T592H T593H T594HT595H T596H T597H T598H T599H T600H T601H T602H T603H T604H T605H T606HT607H T608H (SEQ ID NO: 468) ARDGYGSSSRCLQL T609H T610H T611H T612HT613H T614H T61SH T616H T617H T618H T619H T620H T621H T622H T623H T624HT625H T626H T627H (SEQ ID NO: 468) ARDGYGSSSRCLQL T628H T629H T630HT631H T632H T633H T634H T635H T636H T637H T638H T639H T640H T641H T642HT643H T644H T645H T646H (SEQ ID NO: 468) ARDGYGSSSRCLQL T647H T648HT649H T650H T651H T652H T653H T654H T655H T656H T657H T658H T659H T660HT661H T662H T663H T664H T665H (SEQ ID NO: 468) T0681H CDR1 Mutants CDR1A C D E F G H I K L M N P Q R S T V W Y RYSFTSYY T690H T691H T692H T693HT694H T695H T696H T697H T698H T699H T700H T701H T702H T703H T704H T705HT706H T707H T708H (SEQ ID NO: 462) RYSFTSYY T709H T710H T711H T712HT713H T714H T715H T716H T717H T718H T719H T720H T721H T722H T723H T724HT725H T726H T727H (SEQ ID NO: 462) RYSFTSYY T728H T729H T730H T731HT732H T733H T734H T735H T736H T737H T738H T739H T740H T741H T742H T743HT744H T745H T746H (SEQ ID NO: 462) RYSFTSYY T747H T748H T749H T750HT751H T752H T753H T754H T755H T756H T757H T758H T759H T760H T761H T762HT763H T764H T765H (SEQ ID NO: 462) RYSFTSYY T766H T767H T768H T769HT770H T771H T772H T773H T774H T775H T776H T777H T778H T779H T780H T781HT782H T783H T784H (SEQ ID NO: 462) RYSFTSYY T785H T786H T787H T788HT789H T790H T791H T792H T793H T794H T795H T796H T797H T798H T799H T800HT801H T802H T803H (SEQ ID NO: 462) RYSFTSYY T804H T805H T806H T807HT808H T809H T810H T811H T812H T813H T814H T815H T816H T817H T818H T819HT820H T821H T822H (SEQ ID NO: 462) RYSFTSYY T823H T824H T825H T826HT827H T828H T829H T830H T831H T832H T833H T834H T835H T836H T837H T838HT839H T840H T841H (SEQ ID NO: 462) T0681H CDR2 Mutants CDR2 A C D E F GH I K L M N P Q R S T V W Y INPKSGST T842H T843H T844H T845H T846H T847HT848H T849H T850H T851H T852H T853H T854H T855H T856H T857H T858H T859HT860H (SEQ ID NO: 467) INPKSGST T861H T862H T863H T864H T865H T866HT867H T868H T869H T870H T871H T872H T873H T874H T875H T876H T877H T878HT879H (SEQ ID NO: 467) INPKSGST T880H T881H T882H T883H T884H T885HT886H T887H T888H T889H T890H T891H T892H T893H T894H T895H T896H T897HT898H (SEQ ID NO: 467) INPKSGST T899H T900H T901H T902H T903H T904HT905H T906H T907H T908H T909H T910H T911H T912H T913H T914H T915H T916HT917H (SEQ ID NO: 467) INPKSGST T918H T919H T920H T921H T922H T923HT924H T925H T926H T927H T928H T929H T930H T931H T932H T933H T934H T935HT936H (SEQ ID NO: 467) INPKSGST T937H T938H T939H T940H T941H T942HT943H T944H T945H T946H T947H T948H T949H T950H T951H T952H T953H T954HT955H (SEQ ID NO: 467) INPKSGST T956H T957H T958H T959H T960H T961HT962H T963H T964H T965H T966H T967H T968H T969H T970H T971H T972H T973HT974H (SEQ ID NO: 467) INPKSGST T975H T976H T977H T978H T979H T980HT981H T982H T983H T984H T985H T986H T987H T988H T989H T990H T991H T992HT993H (SEQ ID NO: 467) T678H CDR2 Mutations T678H T850H T925H T926HT951H T958H T989H T990H INPKSGST LNPKSGST INPKIGST INPKKGST INPKSSSTINPKSGDT INPKSGSR INPKSGSS (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID CDR1 Mutations NO: 467) NO: 610) NO: 613)NO: 616) NO: 619) NO: 477) NO: 625) NO: 628) T678HRYSFTSYY (SEQ ID NO: 462) T678H T1015H T1022H T1029H T1036H T1043HT1050H T1057H T713H RFSFTSYY (SEQ ID NO: 592) T1009H T1016H T1023HT1030H T1037H T1044H T1051H T1058H T734H RYHFTSYY (SEQ ID NO: 595)T1010H T1017H T1024H T1031H T1038H T1045H T1052H T1059H T736HRYKFTSYY (SEQ ID NO: 598) T1011H T1018H T1025H T1032H T1039H T1046HT1053H T1060H T742H RYRFTSYY (SEQ ID NO: 601) T1012H T1019H T1026HT1033H T1040H T1047H T1054H T1061H T774H RYSFKSYY (SEQ ID NO: 604)T1013H T1020H T1027H T1034H T1041H T1048H T1055H T1062H T785HRYSFTAYY (SEQ ID NO: 607) T1014H T1021H T1028H T1035H T1042H T1049HT1056H T1063H T681H CDR2 Mutations T678H T850H T925H T926H T951H T958HT989H T990H INPKSGST LNPKSGST INPKIGST INPKKGST INPKSSST INPKSGDTINPKSGSR INPKSGSS (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID CDR1 Mutations NO: 467) NO: 610) NO: 613) NO: 616)NO: 619) NO: 477) NO: 625) NO: 628) T681H RYSFTSYY (SEQ ID NO: 462)T681H T850H T925H T926H T951H T958H T989H T990H T713HRFSFTSYY (SEQ ID NO: 592) T713H T1064H T1070H T1076H T1082H T1088HT1094H T1100H T734H RYHFTSYY (SEQ ID NO: 595) T734H T1065H T1071H T1077HT1083H T1089H T1095H T1101H T736H RYKFTSYY (SEQ ID NO: 598) T736H T1066HT1072H T1078H T1084H T1090H T1096H T1102H T742HRYRFTSYY (SEQ ID NO: 601) T742H T1067H T1073H T1079H T1085H T1091HT1097H T1103H T774H RYSFKSYY (SEQ ID NO: 604) T774H T1068H T1074H T1080HT1086H T1092H T1098H T1104H T785H RYSFTAYY (SEQ ID NO: 607) T785H T1069HT1075H T1081H T1087H T1093H T1099H T1105H T687H CDR2 Mutations T678HT850H T925H T926H T951H T958H T989H T990H INPKSGST LNPKSGST INPKIGSTINPKKGST INPKSSST INPKSGDT INPKSGSR INPKSGSS (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CDR1 Mutations NO: 467) NO: 610)NO: 613) NO: 616) NO: 619) NO: 477) NO: 625) NO: 628) T687HRYSFTSYY (SEQ ID NO: 462) T687H T1113H T1120H T1127H T1134H T1141HT1148H T1155H T713H RFSFTSYY (SEQ ID NO: 592) T1107H T1114H T1121HT1128H T1135H T1142H T1149H T1156H T734H RYHFTSYY (SEQ ID NO: 595)T1108H T1115H T1122H T1129H T1136H T1143H T1150H T1157H T736HRYKFTSYY (SEQ ID NO: 598) T1109H T1116H T1123H T1130H T1137H T1144HT1151H T1158H T742H RYRFTSYY (SEQ ID NO: 601) T1110H T1117H T1124HT1131H T1138H T1145H T1152H T1159H T774H RYSFKSYY (SEQ ID NO: 604)T1111H T1118H T1125H T1132H T1139H T1146H T1153H T1160H T785HRYSFTAYY (SEQ ID NO: 607) T1112H T1119H T1126H T1133H T1140H T1147HT1154H T1161H 

What is claimed is:
 1. A bispecific antibody that binds FIXa and FX andcatalyses FIXa-mediated activation of FX, wherein the antibody comprisestwo immunoglobulin heavy-light chain pairs, wherein a first heavy-lightchain pair comprises a FIXa binding Fv region comprising a first VHdomain paired with a first VL domain, and a second heavy-light chainpair comprises a FX binding Fv region comprising a second VH domainpaired with a second VL domain, wherein the first VH domain comprises aset of HCDRs comprising HCDR1, HCDR2 and HCDR3 with amino acid sequencesdefined wherein HCDR1 is SEQ ID NO: 441; HCDR2 is SEQ ID NO: 436; andHCDR3 is SEQ ID NO: 433, and wherein the first VH domain is at least 95%identical to the N1280H VH domain SEQ ID NO: 443 at the amino acidsequence level; the second VH domain comprises a set of HCDRs comprisingHCDR1, HCDR2 and HCDR3 with amino acid sequences defined wherein HCDR1is SEQ ID NO: 598, SEQ ID NO: 2 is SEQ ID NO: 467 and HCDR3 is SEQ IDNO: 565, and wherein the second VH domain is at least 95% identical tothe T0999H VH domain SEQ ID NO: 632 at the amino acid sequence level,and the first VL domain and the second VL domain each comprise a set ofLCDRs comprising LCDR1, LCDR2 and LCDR3 with amino acid sequencesdefined wherein LCDR1 is SEQ ID NO: 6, LCDR2 is SEQ ID NO: 7 and LCDR3is SEQ ID NO: 8, and wherein the first VL domain and the second VLdomain are at least 95% identical to the 0128L VL domain SEQ ID NO: 10at the amino acid sequence level.
 2. The bispecific antibody accordingto claim 1, wherein the first VII domain is the N1280H VH domain SEQ. IDNO:
 443. 3. The bispecific antibody according to claim 1, wherein thefirst domain is the N441H VII domain SEQ ID NO:
 456. 4. The bispecificantibody according to claim 1, wherein the second VH domain comprisesSEQ ID NO:
 632. 5. The bispecific antibody according to claim 1, whereinthe first VL domain and the second VL domain are identical in amino acidsequence.
 6. The bispecific antibody according to claim 5, wherein thefirst VL domain and the second VL domain comprise the 0325L amino acidsequence SEQ ID NO:
 416. 7. The bispecific antibody according to claim1, wherein each heavy-light chain pair further comprises a CL constantdomain paired with a CH1 domain.
 8. The bispecific antibody according toclaim 1, wherein the heavy-light chain pairs comprise a common lightchain.
 9. The bispecific antibody according to claim 8, wherein thecommon light chain comprises the CL amino acid sequence SEQ ID NO: 146of the 0128L light chain.
 10. The bispecific antibody according to claim9, wherein the common light chain is the 0325L light chain SEQ ID NO:414.
 11. The bispecific antibody according to claim 1, wherein the heavychain of each heavy-light chain comprises a heavy chain constant regionand wherein the first and second heavy-light chain pairs associate toform tetrameric immunoglobulin through dimerisation of the heavy chainconstant regions.
 12. The bispecific antibody according to claim 11,wherein the heavy chain constant region of the first heavy-light chainpair comprises a different amino acid sequence from the heavy chainconstant region of the second heavy-light chain pair, wherein thedifferent amino acid sequences are engineered to promoteheterodimerisation of the heavy chain constant regions.
 13. Thebispecific antibody according to claim 11, wherein the heavy chainconstant region of one or both heavy-light chain pairs is a human IgG4constant region comprising substitution S228P, wherein constant regionnumbering is according to the EU numbering system.
 14. The bispecificantibody according to claim 11, wherein the heavy chain constant regionof one (e.g., the first) heavy-light chain pair comprises SEQ ID NO: 409and the heavy chain constant region of the other (e.g., the second)heavy-light chain pair comprises SEQ ID NO:
 410. 15. The bispecificantibody according to claim 11, comprising a first heavy chaincomprising a first VH domain amino acid sequence SEQ ID NO: 443 or SEQID NO: 456, a second heavy chain comprising a second VH domain aminoacid sequence SEQ ID NO: 632, and a common light chain comprising a VLdomain amino acid sequence SEQ ID NO:
 416. 16. The bispecific antibodyaccording to claim 11, comprising a first heavy chain comprising aminoacid sequence SEQ ID NO: 419, a second heavy chain comprising amino acidsequence SEQ ID NO: 421, and a common light chain comprising amino acidsequence SEQ ID NO:
 414. 17. The bispecific antibody according to claim11, comprising a first heavy chain comprising amino acid sequence SEQ IDNO: 426 a second heavy chain comprising amino acid sequence SEQ ID NO:421, and a common light chain comprising amino acid sequence SEQ ID NO:414.